Compositions and methods of treating lupus nephritis

ABSTRACT

The invention provides methods for treating or delaying progression of lupus nephritis in an individual that has lupus. In some embodiments, the methods comprise administering to the individual an effective amount of a type II anti-CD20 antibody. The invention also provides methods for treating or delaying progression of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in an individual. In some embodiments, the methods comprise administering an effective amount of an anti-CD20 antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 62/159,876, filed May 11, 2015; and 62/300,052,filed Feb. 25, 2016; each of which is incorporated herein by referencein its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392032200SeqList.txt,date recorded: May 5, 2016, size: 37 KB).

FIELD OF THE INVENTION

Provided herein are methods for treating or delaying progression oflupus nephritis in an individual that has lupus by administering a typeII anti-CD20 antibody. Also provided herein are methods for treating ordelaying progression of rheumatoid arthritis (RA) or systemic lupuserythematosus (SLE) in an individual by administering an anti-CD20antibody.

BACKGROUND

Lupus is an autoimmune disease involving antibodies that attackconnective tissue. The disease is estimated to affect nearly 1 millionAmericans, primarily women between the ages of 20-40. The principal formof lupus is a systemic one (systemic lupus erythematosus; SLE). SLE hasan incidence of about 1 in 700 women between the ages of 20 and 60. SLEcan affect any organ system and can cause severe tissue damage.Untreated lupus can be fatal as it progresses from attack of skin andjoints to internal organs, including lung, heart, and kidneys, withrenal disease, termed lupus nephritis (LN), being the primary concern.Lupus mainly appears as a series of flare-ups, with intervening periodsof little or no disease manifestation.

LN is one of the most acute areas of damage associated withpathogenicity in SLE, and accounts for at least 50% of the mortality andmorbidity of the disease. Currently, there are no really curativetreatments for patients who have been diagnosed with SLE or LN. From apractical standpoint, physicians generally employ a number of powerfulimmunosuppressive drugs such as high-dose corticosteroids, e.g.,prednisone, or azathioprine or cyclophosphamide, which are given duringperiods of flare-ups, but may also be given persistently for those whohave experienced frequent flare-ups. Even with effective treatment,which reduces symptoms and prolongs life, many of these drugs havepotentially harmful side effects to the patients being treated. As such,there remains a need for more effective treatments against LN with fewerharmful side effects.

Two anti-CD20 antibodies have been tested in clinical studies forefficacy in treating lupus nephritis. Rituximab, a type I anti-CD20antibody, failed to meet its primary endpoint of overall response(weighted toward complete renal response, or CRR) but resulted in a15.3% increase in partial renal response (PRR) (Rovin, B. H. et al.(2012) Arthritis Rheum. 64:1215-1226). Ocrelizumab, another type Ianti-CD20 antibody, was terminated, in part, because of an imbalance ofserious infectious events (Mysler, E. F. et al. (2013) Arthritis Rheum.65:2368-2379).

Obinutuzumab, a type II anti-CD20 antibody, has been shown to producesuperior B cell depletion, as compared to rituximab. Significantlygreater B cell depletion was observed with obinutuzumab treatment,compared to rituximab treatment, in cynomolgous monkeys (Mossner, E. etal. (2010) Blood 115:4393-4402). Therefore, there remains a need fortesting the efficacy of type II anti-CD20 antibodies in treating orpreventing LN in patients with lupus.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY

In certain aspects, provided herein are methods for treating or delayingprogression of lupus nephritis in an individual, comprisingadministering to the individual at least a first antibody exposure to atype II anti-CD20 antibody and a second antibody exposure to the type IIanti-CD20 antibody. In some embodiments, the individual has lupus. Insome embodiments, the second antibody exposure is not provided untilfrom about 18 weeks to about 26 weeks after the first antibody exposure.In some embodiments, the second antibody exposure is not provided untilfrom about 4.5 months to about 6.5 months after the first antibodyexposure. In some embodiments, the first antibody exposure comprises oneor two doses of the type II anti-CD20 antibody, the first antibodyexposure comprising a total exposure of between about 1800 mg and about2200 mg of the type II anti-CD20 antibody. In some embodiments, thesecond antibody exposure comprises one or two doses of the type IIanti-CD20 antibody, the second antibody exposure comprising a totalexposure of between about 1800 mg and about 2200 mg of the type IIanti-CD20 antibody. In some embodiments, the type II anti-CD20 antibodycomprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:1,HVR-H2 sequence of SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, anda light chain comprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequenceof SEQ ID NO:5, and HVR-L3 sequence of SEQ ID NO:6. In some embodiments,the individual is at risk for developing class III or class IV lupusnephritis. In some embodiments, the methods are for preventing lupusnephritis in an individual that has lupus. In some embodiments, themethods are for preventing lupus nephritis in an individual that hasSLE. In some embodiments, the methods are for treating or delayingprogression of lupus nephritis in an individual that has SLE.

In some embodiments, the first antibody exposure comprises a first doseof the type II anti-CD20 antibody and a second dose of the type IIanti-CD20 antibody, and the second dose of the first antibody exposureis not provided until from about 1.5 weeks to about 2.5 weeks after thefirst dose of the first antibody exposure. In some embodiments, thefirst antibody exposure comprises a first dose of the type II anti-CD20antibody and a second dose of the type II anti-CD20 antibody, and thesecond dose of the first antibody exposure is not provided until about 2weeks after the first dose of the first antibody exposure. In someembodiments, the first antibody exposure comprises a first dose of thetype II anti-CD20 antibody and a second dose of the type II anti-CD20antibody, and the second dose of the first antibody exposure is notprovided until from about 10 days to about 17 days after the first doseof the first antibody exposure. In some embodiments, the first antibodyexposure comprises a first dose of the type II anti-CD20 antibody and asecond dose of the type II anti-CD20 antibody, and the second dose ofthe first antibody exposure is not provided until about 14 after thefirst dose of the first antibody exposure. In some embodiments, thefirst dose of the first antibody exposure is about 1000 mg of the typeII anti-CD20 antibody. In some embodiments, the second dose of the firstantibody exposure is about 1000 mg of the type II anti-CD20 antibody. Insome embodiments, the second antibody exposure comprises a first dose ofbetween about 900 mg and about 1100 mg of the type II anti-CD20 antibodyand a second dose of between about 900 mg and about 1100 mg of the typeII anti-CD20 antibody. In some embodiments, the second antibody exposurecomprises a first dose of the type II anti-CD20 antibody and a seconddose of the type II anti-CD20 antibody, and the second dose of thesecond antibody exposure is not provided until from about 1.5 weeks toabout 2.5 weeks after the first dose of the second antibody exposure. Insome embodiments, the second antibody exposure comprises a first dose ofthe type II anti-CD20 antibody and a second dose of the type IIanti-CD20 antibody, and the second dose of the second antibody exposureis not provided until about 2 weeks after the first dose of the secondantibody exposure. In some embodiments, the second antibody exposurecomprises a first dose of the type II anti-CD20 antibody and a seconddose of the type II anti-CD20 antibody, and the second dose of thesecond antibody exposure is not provided until from about 10 days toabout 17 days after the first dose of the second antibody exposure. Insome embodiments, the second antibody exposure comprises a first dose ofthe type II anti-CD20 antibody and a second dose of the type IIanti-CD20 antibody, and the second dose of the second antibody exposureis not provided until about 14 days after the first dose of the secondantibody exposure. In some embodiments, the first dose of the secondantibody exposure is about 1000 mg of the type II anti-CD20 antibody. Insome embodiments, the second dose of the second antibody exposure isabout 1000 mg of the type II anti-CD20 antibody. In some embodiments,the first antibody exposure and the second antibody exposure areadministered intravenously. In some embodiments, the individual hasclass III or class IV lupus nephritis. In some embodiments, theindividual is at risk for developing class III or class IV lupusnephritis.

In certain aspects, provided herein are methods for treating or delayingprogression of lupus nephritis in an individual that has lupus,comprising administering to the individual an effective amount of a typeII anti-CD20 antibody; wherein the type II anti-CD20 antibody comprisesa heavy chain comprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequenceof SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chaincomprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ IDNO:5, and HVR-L3 sequence of SEQ ID NO:6; and wherein the individual hasclass III or class IV lupus nephritis. In some embodiments, theindividual is at risk for developing class III or class IV lupusnephritis. In some embodiments, the methods are for preventing lupusnephritis in an individual that has lupus. In some embodiments, themethods are for preventing lupus nephritis in an individual that hasSLE. In some embodiments, the methods are for treating or delayingprogression of lupus nephritis in an individual that has SLE.

In certain aspects, provided herein are methods for treating or delayingprogression of lupus nephritis in an individual that has lupus,comprising administering to the individual a dose of about 1000 mg of atype II anti-CD20 antibody, wherein the type II anti-CD20 antibodycomprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:1,HVR-H2 sequence of SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, anda light chain comprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequenceof SEQ ID NO:5, and HVR-L3 sequence of SEQ ID NO:6, and wherein the doseis administered to the individual once on days 1, 15, 168, and 182. Incertain aspects, provided herein are methods for treating or delayingprogression of lupus nephritis in an individual that has lupus,comprising administering to the individual a dose of about 1000 mg of atype II anti-CD20 antibody, wherein the type II anti-CD20 antibodycomprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:1,HVR-H2 sequence of SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, anda light chain comprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequenceof SEQ ID NO:5, and HVR-L3 sequence of SEQ ID NO:6, and wherein the doseis administered to the individual once on weeks 0, 2, 24, and 26. Insome embodiments, week 0 corresponds to day 1. In some embodiments, theindividual has class III or class IV lupus nephritis. In someembodiments, the type II anti-CD20 antibody is obinutuzumab.

In some embodiments of any of the above embodiments, the type IIanti-CD20 antibody is administered intravenously. In some embodiments ofany of the above embodiments, the individual does not have class III (C)or class IV (C) lupus nephritis. In some embodiments of any of the aboveembodiments, the individual has class V lupus nephritis. In someembodiments of any of the above embodiments, the methods further includeadministering to the individual an effective amount of animmunosuppressive agent. In some embodiments, the immunosuppressiveagent comprises mycophenolic acid, a derivative thereof, or a saltthereof. In some embodiments, the immunosuppressive agent comprisesmycophenolate mofetil. In some embodiments of any of the aboveembodiments, the methods further include administering to the individualan effective amount of a glucocorticoid or corticosteroid. In someembodiments, the glucocorticoid or corticosteroid comprisesmethylprednisolone. In some embodiments, the glucocorticoid orcorticosteroid comprises prednisone. In some embodiments of any of theabove embodiments, the methods further include administering to theindividual an effective amount of an antihistamine. In some embodiments,the antihistamine comprises diphenhydramine. In some embodiments of anyof the above embodiments, the methods further include administering tothe individual an effective amount of a non-steroidal anti-inflammatorydrug (NSAID). In some embodiments, the NSAID comprises acetaminophen. Insome embodiments of any of the above embodiments, the methods furtherinclude administering to the individual a standard of care treatment. Insome embodiments, the standard of care treatment comprises treatmentwith one or more of an angiotensin-converting enzyme (ACE) inhibitor, anangiotensin-receptor blocker, cyclophosphamide, mycophenolate mofetil,azathioprine, and a glucocorticoid or corticosteroid. In someembodiments, the standard of care treatment is administered after thefirst antibody exposure to the type II anti-CD20 antibody and/or afterthe second antibody exposure to the type II anti-CD20 antibody. In someembodiments of any of the above embodiments, the methods further includeadministering to the individual an effective amount of anantihypertensive agent. In some embodiments, the antihypertensive agentis an angiotensin-converting enzyme (ACE) inhibitor or anangiotensin-receptor blocker. In some embodiments of any of the aboveembodiments, the method results in a complete renal response (CRR) inthe individual. In some embodiments of any of the above embodiments, themethod results in a depletion of circulating peripheral B cells in theindividual. In some embodiments, the circulating peripheral B cells areCD19+ B cells. In some embodiments of any of the above embodiments, thetype II anti-CD20 antibody is a humanized or human antibody. In someembodiments of any of the above embodiments, the type II anti-CD20antibody is afucosylated. In some embodiments of any of the aboveembodiments, the type II anti-CD20 antibody is nonfucosylated (e.g., asdescribed in U.S. Pat. No. 8,883,980). In some embodiments of any of theabove embodiments, the heavy chain of the type II anti-CD20 antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:7. In some embodiments of any of the aboveembodiments, the light chain of the type II anti-CD20 antibody comprisesa light chain variable region comprising the amino acid sequence of SEQID NO:8. In some embodiments of any of the above embodiments, the typeII anti-CD20 antibody is obinutuzumab. In some embodiments of any of theabove embodiments, the individual or patient is a human.

In certain aspects, provided herein are kits or articles of manufacturefor treating or delaying progression of lupus nephritis in an individualthat has lupus, comprising (a) a container comprising a type IIanti-CD20 antibody, wherein the type II anti-CD20 antibody comprises aheavy chain comprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequenceof SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chaincomprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ IDNO:5, and HVR-L3 sequence of SEQ ID NO:6; and (b) a package insert withinstructions for treating or delaying progression of lupus nephritis inan individual, wherein the instructions indicate that at least a firstantibody exposure to a type II anti-CD20 antibody and a second antibodyexposure to the type II anti-CD20 antibody are administered to theindividual, the second antibody exposure not being provided until fromabout 18 weeks to about 26 weeks after the first antibody exposure;wherein the first antibody exposure comprises one or two doses of thetype II anti-CD20 antibody, the first antibody exposure comprising atotal exposure of between about 1800 mg and about 2200 mg of the type IIanti-CD20 antibody; and wherein the second antibody exposure comprisesone or two doses of the type II anti-CD20 antibody, the second antibodyexposure comprising a total exposure of between about 1800 mg and about2200 mg of the type II anti-CD20 antibody. In some embodiments, the kitsor articles of manufacture further include (c) a second medicament,wherein the type II anti-CD20 antibody is a first medicament; and (d)instructions on the package insert for administering the secondmedicament to the subject. In some embodiments, the second medicament isan immunosuppressive agent, a glucocorticoid, a corticosteroid, ananti-malarial agent, a cytotoxic agent, an integrin antagonist, acytokine antagonist, or a hormone. In some embodiments, the heavy chainof the type II anti-CD20 antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:7. In someembodiments, the light chain of the type II anti-CD20 antibody comprisesa light chain variable region comprising the amino acid sequence of SEQID NO:8. In some embodiments, the type II anti-CD20 antibody isobinutuzumab. In some embodiments, the kits or articles of manufactureare for preventing lupus nephritis in an individual that has SLE. Insome embodiments, the kits or articles of manufacture are for treatingor delaying progression of lupus nephritis in an individual that hasSLE.

In certain aspects, provided herein are methods for treating or delayingprogression of rheumatoid arthritis (RA) or systemic lupus erythematosus(SLE) in an individual, comprising administering to the individual aneffective amount of an anti-CD20 antibody, wherein the antibodycomprises a heavy chain variable region comprising an HVR-H1 sequence ofSEQ ID NO:1, an HVR-H2 sequence of SEQ ID NO:2, and an HVR-H3 sequenceof SEQ ID NO:3, and a light chain variable region comprising an HVR-L1sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and anHVR-L3 sequence of SEQ ID NO:6. In some embodiments, the antibody isadministered intravenously. In some embodiments, the method results in adepletion of circulating peripheral B cells in the individual. In someembodiments, the circulating peripheral B cells are CD19+ B cells. Insome embodiments, the antibody is a humanized or human antibody. In someembodiments, the antibody is afucosylated. In some embodiments, theheavy chain variable region comprises the amino acid sequence of SEQ IDNO:7. In some embodiments, the light chain variable region comprises theamino acid sequence of SEQ ID NO:8. In some embodiments, the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO:7 and thelight chain variable region comprises the amino acid sequence of SEQ IDNO:8. In some embodiments, the antibody is obinutuzumab. In someembodiments, the antibody comprises a modified Fc region. In someembodiments, the Fc region comprises a modification for attenuatingeffector function. In some embodiments, the Fc region is a human IgG1 Fcregion. In some embodiments, the human IgG1 Fc region comprises L234A,L235A and P329G amino acid substitutions, numbering according to EUindex.

In certain aspects, provided herein are compositions for use in treatingor delaying progression of rheumatoid arthritis (RA) or systemic lupuserythematosus (SLE) in an individual, the compositions comprising ananti-CD20 antibody, wherein the antibody comprises a heavy chainvariable region comprising an HVR-H1 sequence of SEQ ID NO:1, an HVR-H2sequence of SEQ ID NO:2, and an HVR-H3 sequence of SEQ ID NO:3, and alight chain variable region comprising an HVR-L1 sequence of SEQ IDNO:4, an HVR-L2 sequence of SEQ ID NO:5, and an HVR-L3 sequence of SEQID NO:6. In some embodiments, the composition is administeredintravenously. In some embodiments, administering the compositionresults in a depletion of circulating peripheral B cells in theindividual. In some embodiments, the circulating peripheral B cells areCD19+ B cells. In some embodiments, the antibody is a humanized or humanantibody. In some embodiments, the antibody is afucosylated. In someembodiments, the heavy chain variable region comprises the amino acidsequence of SEQ ID NO:7. In some embodiments, the light chain variableregion comprises the amino acid sequence of SEQ ID NO:8. In someembodiments, the heavy chain variable region comprises the amino acidsequence of SEQ ID NO:7 and the light chain variable region comprisesthe amino acid sequence of SEQ ID NO:8. In some embodiments, theantibody is obinutuzumab. In some embodiments, the antibody comprises amodified Fc region. In some embodiments, the Fc region comprises amodification for attenuating effector function. In some embodiments, theFc region is a human IgG1 Fc region. In some embodiments, the human IgG1Fc region comprises L234A, L235A and P329G amino acid substitutions,numbering according to EU index. In some embodiments of any of the aboveembodiments, the individual or patient is a human.

In certain aspects, provided herein is use of an anti-CD20 antibody forthe manufacture of a medicament for use in treatment of rheumatoidarthritis (RA) or systemic lupus erythematosus (SLE) in an individual,wherein the antibody comprises a heavy chain variable region comprisingan HVR-H1 sequence of SEQ ID NO:1, an HVR-H2 sequence of SEQ ID NO:2,and an HVR-H3 sequence of SEQ ID NO:3, and a light chain variable regioncomprising an HVR-L1 sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQID NO:5, and an HVR-L3 sequence of SEQ ID NO:6.

In certain aspects, provided herein are kits or articles of manufacturefor treating or delaying progression of rheumatoid arthritis (RA) orsystemic lupus erythematosus (SLE) in an individual, comprising (a) acontainer comprising an anti-CD20 antibody, wherein the antibodycomprises a heavy chain variable region comprising an HVR-H1 sequence ofSEQ ID NO:1, an HVR-H2 sequence of SEQ ID NO:2, and an HVR-H3 sequenceof SEQ ID NO:3, and a light chain variable region comprising an HVR-L1sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and anHVR-L3 sequence of SEQ ID NO:6; and (b) a package insert withinstructions for administering an effective amount of anti-CD20 antibodyto treat or delay progression of rheumatoid arthritis (RA) or systemiclupus erythematosus (SLE) in an individual. In some embodiments, thepackage insert includes instructions for administering the antibodyintravenously. In some embodiments, the antibody is a humanized or humanantibody. In some embodiments, the antibody is afucosylated. In someembodiments, the heavy chain variable region comprises the amino acidsequence of SEQ ID NO:7. In some embodiments, the light chain variableregion comprises the amino acid sequence of SEQ ID NO:8. In someembodiments, the heavy chain variable region comprises the amino acidsequence of SEQ ID NO:7 and the light chain variable region comprisesthe amino acid sequence of SEQ ID NO:8. In some embodiments, theantibody is obinutuzumab. In some embodiments, the antibody comprises amodified Fc region. In some embodiments, the Fc region comprises amodification for attenuating effector function. In some embodiments, theFc region is a human IgG1 Fc region. In some embodiments, the human IgG1Fc region comprises L234A, L235A and P329G amino acid substitutions,numbering according to EU index.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the study design for a Phase II study examiningobinutuzumab+mycophenolate mofetil vs. placebo+mycophenolate mofetil.EP=endpoint; MMF=mycophenolate mofetil.

FIG. 2A-2D show whole blood B-cell-depletion, internalization andcomplement-dependent cellular cytotoxicity elicited by Obinutuzumab orRituximab in RA and SLE patient samples. FIG. 2A shows whole bloodsamples from patients with RA (n=31) and SLE (n=34). Samples wereincubated with or without anti-CD20 mAbs, RTX, OBZ_(Gly) and OBZ for 24hours before flow cytometry to analyze B cell death. Values are the meanof triplicate wells. The horizontal line in the box represents themedian, the box represents the interquartile range and the whiskersrepresent the range. FIG. 2B shows the frequency of surface accessiblemAbs. The frequency was assessed by flow cytometry after six hours ofincubation with isolated B cells from patients with RA (n=5) and SLE(n=8) with or without prior incubation with anti-FcγRII blocking mAb,AT10. The horizontal line represents the median. FIG. 2C shows CDCinduced by RTX and OBZ. Isolated B cells (Healthy control (HC), n=2; RA,n=2 and SLE, n=3) were incubated with RTX or OBZ for 30 minutes withnormal healthy serum (NHS) or heat inactivated serum (HIS) beforeanalyzing for the frequency of lysed CD19+Av+PI+ B cells. FIG. 2D showsthe fold increase in CD19+Av+PI+ cells in samples incubated with NHS vsHIS representing the efficiency of CDC by mAbs. RTX, rituximab; OBZ,Obinutuzumab; RA, rheumatoid arthritis; SLE, systemic lupuserythematosus. HC, Healthy control * p<0.05; **, p<0.005; ***, p<0.0001and ns, not significant.

FIG. 3A-3G show the flow cytometry-gating strategy to assess NK celldegranulation, describing the relationship between NK cell expression ofCD107a and CD16. Whole blood samples were incubated with or without mAbsfor 24 hours before analyzing by flow cytometry. NK cells wereidentified based on forward- and side-scatter properties and expressionof CD56 but not CD3. The frequency of CD3-CD56+CD107a+ cells representedactivated/degranulated NK cells. FSC, forward-scatter; SSC,side-scatter. FIG. 3A shows flow cytometry gating of forward scatter vs.side scatter. FIG. 3B shows flow cytometry gating of CD56 vs. CD3. FIG.3C shows flow cytometry gating of forward scatter vs. CD107a. FIG. 3Dshows flow cytometry gating of forward scatter vs. CD16. Threesubpopulations of CD3-CD56+ NK cells were identified based on therelative expression of CD16 (boxed as high, medium, and low). Therelative frequency of activated CD107a+NK cells differed in these 3subpopulations based on CD16 expression in a hierarchical mannerCD16++<CD16+<CD16−. FIG. 3E shows flow cytometry gating of forwardscatter vs. CD107a for the high box. FIG. 3F shows flow cytometry gatingof forward scatter vs. CD107a for the medium box. FIG. 3G shows flowcytometry gating of forward scatter vs. CD107a for the low box.

FIG. 4A-4D show that OBZ is more efficient than RTX at activating NKcells in RA and SLE patient samples. NK cell activation was assessed inwhole blood samples from patients with RA (n=18) and SLE (n=23)incubated for 24 hours in the presence or absence of mAbs. *, p<0.05;**, p<0.005; ***, p<0.0001; ns, not significant and Spearman correlationcoefficient, r² was considered significant when p was at least <0.05.FIG. 4A shows the frequency of CD3-CD56+NK cells in the lymphocyte gate,CD3-CD56+CD107a+NK cells and CD3-CD56-CD16+NK cells as a percentage oftotal NK cells or CD19+ cells. Horizontal lines represent the median.FIG. 4B shows the frequency of CD3-CD56+CD107a+NK cells and the foldincrease in the frequency of CD3-CD56+CD107a+NK cells and the frequencyof CD3−CD56+16+NK cells in samples incubated with RTX and OBZ, frompatients with RA and SLE. Horizontal lines represent the median. FIG. 4Cshows the relationship between the frequency of CD3-CD56+CD107a+NK cellsin samples incubated with or without RTX and OBZ in samples frompatients with RA (n=18). FIG. 4D shows the relationship between thefrequency of CD3-CD56+CD107a+NK cells in samples incubated with orwithout RTX and OBZ in samples from patients with SLE (n=23).

FIG. 5A-5D show that Obinutuzumab is more efficient than Rituximab atevoking NK cell-mediated cellular cytotoxicity in RA and SLE patientsamples. FIG. 5A shows a whole blood B-cell depletion assay showing thepercentage B-cell depletion by RTX, OBZ_(Gly) and OBZ in samples frompatients with RA (n=18) and SLE (n=23). Box and whiskers represent theinterquartile range and the range, the horizontal line in the boxrepresents the median. FIG. 5B shows the frequency of CD3-CD56+CD107a+NKcells in whole blood samples from patients with RA and SLE after 24-hourincubation with or without mAbs, analyzed by flow cytometry. FIG. 5Cshows the relative increase in the frequency of CD3-CD56+CD107a+NK cellsin whole blood samples incubated with or without mAbs, analyzed by flowcytometry. FIG. 5D shows the frequency of CD3-CD56+CD16+NK cells inwhole blood samples from patients with RA (n=18) and SLE (n=23) after 24hour incubation with or without mAbs, analyzed by flow cytometry. Forthe bar graphs, the error bars represent the median and interquartileranges. * p<0.05; **, p<0.005; ***, p<0.0001; and ns, not significant.

FIG. 6A-6D show that Obinutuzumab is more efficient than Rituximab atactivating neutrophils in RA and SLE patient samples. FIG. 6A shows themean fluorescence intensity (MFI) of CD11b on CD15+ neutrophils after 24hour incubation of whole blood samples from patients with RA (n=10) andSLE (n=22) incubated with or without mAbs (1 μg/ml). The Median andinterquartile ranges are represented by the error bars. FIG. 6B showsthe relationship between the MFI of CD11b on CD15+neutrophils in samplesincubated with or without mAbs in RA and SLE samples. FIG. 6C shows theMFI of CD62L on CD15+neutrophils in samples incubated with or withoutmAbs in RA and SLE samples. FIG. 6D shows the relationship between theMFI of CD62L on CD15+ neutrophils, in samples incubated with or withoutmAbs in RA (n=10) and SLE (n=22) samples. * p<0.05; **, p<0.005; ***,p<0.0001. Spearman correlation coefficient, r² was consideredsignificant when p was at least <0.05.

FIG. 7A-7D show assessment of direct cell death, internalization andexpression of CD20 and FcγRIIb in B-cell subpopulations from RA and SLEsamples. FIG. 7A shows the frequency of Annexin V+ cells as a proportionof all CD19+ B cells and also B-cell subpopulations based on therelative expression of IgD and CD27: (IgD+CD27-naïve cells; IgD+CD27+unswitched memory cells; IgD-CD27+ switched memory cells; andIgD-CD27-double negative cells); in samples from patients with RA (n=5)and SLE (n=4) incubated with or without mAbs. FIG. 7B shows the meanfluorescence intensity (MFI) of CD20 on all CD19+ cells and B-cellsubpopulations in samples from patients with SLE (n=9). FIG. 7C shows asurface fluorescence-quenching assay. The frequency of surfaceaccessible mAbs after 6 hours of incubation with isolated B-cells, withor without prior incubation with anti-FcγRII mAb, AT10 from patientswith SLE (n=9), in all CD19+ B-cells and B-cell subpopulations. FIG. 7Dshows the MFI of FcγRIIb on all CD19+ B-cells and B-cell subpopulationsin samples from patients with SLE (n=9). For the bar graphs, the errorbars represent the median and interquartile ranges. Box and whiskersrepresent the interquartile range and the horizontal line in the boxrepresents the median. * p<0.05; **, p<0.005; ***, p<0.0001.

FIG. 8 shows the gating strategy for the complement-dependentcytotoxicity assay, and CDC by RTX and OBZ. Isolated B cells wereincubated with mAbs either with NHS or HIS for 30 minutes at roomtemperature before analyzing by flow cytometry. The frequency of AnV+PI+ cells represented cell death. HIS, heat inactivated serum; NHS,normal healthy serum; RTX, rituximab; OBZ, Obinutuzumab; An V, Annexin Vand PI, propidium iodide.

FIG. 9 shows the flow cytometry-gating strategy to assess neutrophilactivation. After 24 hours of incubation, whole blood samples wereanalysed by flow cytometry. Neutrophils were identified by forward- andside-scatter and CD15 positively. The mean fluorescence intensity ofCD11b and CD62L was analyzed on gated neutrophils positive for CD15.

FIG. 10 shows the flow cytometry-gating strategy to assess direct celldeath. After 6 hours of incubation with or without mAbs at 37° C. and 5%CO₂, isolated B-cells were analyzed by flow cytometry. CD19+ B-cellswere categorized into naïve (IgD+CD27−), unswitched memory cells(IgD+CD27+), switched memory cells (IgD-CD27+) and double negative cells(IgD-CD27−). The frequency of Annexin V+ cells represented direct celldeath.

FIG. 11 shows the inherent susceptibility to spontaneous cell death inB-cell subpopulations. Isolated B-cells incubated in RPMI supplementedwith 10% foetal calf serum for 6 hours at at 37° C. and 5% CO₂ wereanalyzed by flow cytometry. The frequency of Annexin V+ cellsrepresented direct cell death in CD19+ cells as a whole and also inB-cell subpopulations categorized into naïve (IgD+CD27−), unswitchedmemory cells (IgD+CD27+), switched memory cells (IgD-CD27+) and doublenegative cells (IgD-CD27−). Av, Annexin V; * p<0.05; ***, p<0.0001; ns,not significant.

DETAILED DESCRIPTION

In one aspect, provided herein are methods for treating or delayingprogression of lupus nephritis in an individual, including administeringto the individual at least a first antibody exposure to a type IIanti-CD20 antibody and a second antibody exposure to the type IIanti-CD20 antibody. In some embodiments, the individual has lupus. Insome embodiments, the second antibody exposure is not provided untilfrom about 18 weeks to about 26 weeks after the first antibody exposure.In some embodiments, the first antibody exposure includes one or twodoses of the type II anti-CD20 antibody, the first antibody exposurecontaining a total exposure of between about 1800 mg and about 2200 mgof the type II anti-CD20 antibody. In some embodiments, the secondantibody exposure includes one or two doses of the type II anti-CD20antibody, the second antibody exposure containing a total exposure ofbetween about 1800 mg and about 2200 mg of the type II anti-CD20antibody. In some embodiments, the antibody comprises a heavy chaincomprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ IDNO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chain comprisingHVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ ID NO:5, andHVR-L3 sequence of SEQ ID NO:6.

In another aspect, provided herein are methods for treating or delayingprogression of lupus nephritis in an individual that has lupus,including administering to the individual an effective amount of a typeII anti-CD20 antibody. In some embodiments, the antibody includes aheavy chain containing HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequenceof SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chaincontaining HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ IDNO:5, and HVR-L3 sequence of SEQ ID NO:6. In some embodiments, theindividual has class III or class IV lupus nephritis.

In another aspect, provided herein are methods for treating or delayingprogression of rheumatoid arthritis (RA) or systemic lupus erythematosus(SLE) in an individual, comprising administering to the individual aneffective amount of an anti-CD20 antibody. In some embodiments, theantibody comprises a heavy chain variable region comprising an HVR-H1sequence of SEQ ID NO:1, an HVR-H2 sequence of SEQ ID NO:2, and anHVR-H3 sequence of SEQ ID NO:3, and a light chain variable regioncomprising an HVR-L1 sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQID NO:5, and an HVR-L3 sequence of SEQ ID NO:6.

I. GENERAL TECHNIQUES

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J. B. LippincottCompany, 1993).

II. DEFINITIONS

The term “lupus nephritis (LN)” refers to a manifestation of lupus(e.g., systemic lupus erythematosus, drug-induced lupus, neonatal lupus,or discoid lupus) in the kidney(s).

The term “antibody” includes monoclonal antibodies (including fulllength antibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, multispecific antibodies(e.g., bispecific antibodies, diabodies, and single-chain molecules, aswell as antibody fragments (e.g., Fab, F(ab′)₂, and Fv). The term“immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see e.g., Basic and Clinical Immunology, 8th Edition, DanielP. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange,Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains. Depending on the amino acid sequence of the constantdomain of their heavy chains (CH), immunoglobulins can be assigned todifferent classes or isotypes. There are five classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chainsdesignated α, δ, ε, γ and μ, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “VH” and “VL”, respectively. These domains are generally the mostvariable parts of the antibody (relative to other antibodies of the sameclass) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, it is concentrated in three segments calledhypervariable regions (HVRs) both in the light-chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three HVRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The HVRs in each chain are held together in close proximity by the FRregions and, with the HVRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. In contrast topolyclonal antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler andMilstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2^(nd) ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhuet al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and technologies for producing human or human-likeantibodies in animals that have parts or all of the human immunoglobulinloci or genes encoding human immunoglobulin sequences (see, e.g., WO1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits etal., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); andLonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The term “naked antibody” refers to an antibody that is not conjugatedto a cytotoxic moiety or radiolabel.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antibody fragment. Specifically wholeantibodies include those with heavy and light chains including an Fcregion. The constant domains may be native sequence constant domains(e.g., human native sequence constant domains) or amino acid sequencevariants thereof. In some cases, the intact antibody may have one ormore effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments. Papain digestion of antibodies produced twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (V_(H)), and thefirst constant domain of one heavy chain (C_(H)1). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having different antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having a few additional residues at the carboxy terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Functional fragments” of the antibodies of the invention comprise aportion of an intact antibody, generally including the antigen bindingor variable region of the intact antibody or the Fc region of anantibody which retains or has modified FcR binding capability. Examplesof antibody fragments include linear antibody, single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)). Chimeric antibodies of interest herein include PRIMATIZED®antibodies wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by, e.g., immunizing macaque monkeys with anantigen of interest. As used herein, “humanized antibody” is used asubset of “chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR(hereinafter defined) of the recipient are replaced by residues from anHVR of a non-human species (donor antibody) such as mouse, rat, rabbitor non-human primate having the desired specificity, affinity, and/orcapacity. In some instances, framework (“FR”) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, etc. The number of these aminoacid substitutions in the FR are typically no more than 6 in the Hchain, and in the L chain, no more than 3. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is an antibody that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

A “human consensus framework” or “acceptor human framework” is aframework that represents the most commonly occurring amino acidresidues in a selection of human immunoglobulin VL or VH frameworksequences. Generally, the selection of human immunoglobulin VL or VHsequences is from a subgroup of variable domain sequences. Generally,the subgroup of sequences is a subgroup as in Kabat et al., Sequences ofProteins of Immunological Interest, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Examples includefor the VL, the subgroup may be subgroup kappa I, kappa II, kappa III orkappa IV as in Kabat et al., supra. Additionally, for the VH, thesubgroup may be subgroup I, subgroup II, or subgroup III as in Kabat etal., supra. Alternatively, a human consensus framework can be derivedfrom the above in which particular residues, such as when a humanframework residue is selected based on its homology to the donorframework by aligning the donor framework sequence with a collection ofvarious human framework sequences. An acceptor human framework “derivedfrom” a human immunoglobulin framework or a human consensus frameworkmay comprise the same amino acid sequence thereof, or it may containpre-existing amino acid sequence changes. In some embodiments, thenumber of pre-existing amino acid changes are 10 or less, 9 or less, 8or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 orless.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, the VH subgroup III consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (HC-FR1)(SEQID NO:35), WVRQAPGKGLEWV (HC-FR2), (SEQ ID NO:36),RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (HC-FR3, SEQ ID NO:37), WGQGTLVTVSA(HC-FR4), (SEQ ID NO:38).

A “VL kappa I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: DIQMTQSPSSLSASVGDRVTITC (LC-FR1) (SEQID NO:39), WYQQKPGKAPKLLIY (LC-FR2) (SEQ ID NO:40),GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (LC-FR3)(SEQ ID NO:41), FGQGTKVEIKR(LC-FR4)(SEQ ID NO:42).

An “amino-acid modification” at a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specified residue,or the insertion of at least one amino acid residue adjacent thespecified residue. Insertion “adjacent” to a specified residue meansinsertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue. The preferred aminoacid modification herein is a substitution.

An “affinity-matured” antibody is one with one or more alterations inone or more HVRs thereof that result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody that does notpossess those alteration(s). In one embodiment, an affinity-maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity-matured antibodies are produced by procedures known inthe art. For example, Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by VH- and VL-domain shuffling. Randommutagenesis of HVR and/or framework residues is described by, forexample: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994);Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

As use herein, the term “specifically binds to” or is “specific for”refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, which is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody thatspecifically binds to a target (which can be an epitope) is an antibodythat binds this target with greater affinity, avidity, more readily,and/or with greater duration than it binds to other targets. In oneembodiment, the extent of binding of an antibody to an unrelated targetis less than about 10% of the binding of the antibody to the target asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that specifically binds to a target has a dissociation constant(Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In certainembodiments, an antibody specifically binds to an epitope on a proteinthat is conserved among the protein from different species. In anotherembodiment, specific binding can include, but does not require exclusivebinding.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG1, IgG2 (IgG2A,IgG2B), IgG3 and IgG4.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see M. Daeron, Annu.Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J.Immunol. 24: 249 (1994). Methods of measuring binding to FcRn are known(see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997);Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton etal., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004/92219 (Hinton etal.). Binding to FcRn in vivo and serum half-life of human FcRnhigh-affinity binding polypeptides can be assayed, e.g., in transgenicmice or transfected human cell lines expressing human FcRn, or inprimates to which the polypeptides having a variant Fc region areadministered. WO 2004/42072 (Presta) describes antibody variants whichimproved or diminished binding to FcRs. See also, e.g., Shields et al.,J. Biol. Chem. 9(2): 6591-6604 (2001).

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

A “package insert” refers to instructions customarily included incommercial packages of medicaments that contain information about theindications customarily included in commercial packages of medicamentsthat contain information about the indications, usage, dosage,administration, contraindications, other medicaments to be combined withthe packaged product, and/or warnings concerning the use of suchmedicaments, etc.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. For example, an individual is successfully “treated” if oneor more symptoms associated with lupus nephritis are mitigated oreliminated, including, but are not limited to, elevated serumcreatinine, proteinuria, red cell casts, reduced renal function,nephrotic syndrome, granular casts, microhematuria, macrohematuria,hypertension, tubular abnormalities, hyperkalemia, rapidly progressiveglomerulonephritis (RPGN), and acute renal failure (ARF).

As used herein, “delaying progression” of a disease (e.g., lupusnephritis) means to defer, hinder, slow, retard, stabilize, and/orpostpone development of the disease. This delay can be of varyinglengths of time, depending on the history of the disease and/orindividual being treated. As is evident to one skilled in the art, asufficient or significant delay can, in effect, encompass prevention, inthat the individual, e.g., an individual at risk for developing thedisease, does not develop the disease. For example, the progression ofSLE in an individual before the onset of LN symptoms and/or pathologymay be delayed such that the development of LN is postponed orprevented.

As used herein, “complete renal response (CRR)” refers to a response totreatment that includes a normalization of serum creatinine, inactiveurinary sediment, and a urinary protein to creatinine ratio of less than0.5.

As used herein, “partial renal response (PRR)” refers to a response totreatment that is less than a CRR but still includes mitigation of oneor more symptoms including without limitation a reduction in serumcreatinine, reduced urinary sediment, and a reduction in proteinuria.

An “effective amount” is at least the minimum concentration required toeffect a measurable improvement or prevention of a particular disorder.An effective amount herein may vary according to factors such as thedisease state, age, sex, and weight of the patient, and the ability ofthe antibody to elicit a desired response in the individual. Aneffective amount is also one in which any toxic or detrimental effectsof the treatment are outweighed by the therapeutically beneficialeffects. For prophylactic use, beneficial or desired results includeresults such as eliminating or reducing the risk, lessening theseverity, or delaying the onset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as decreasing one or more symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, enhancing effect of another medicationsuch as via targeting, delaying the progression of the disease, and/orprolonging survival. In the case of lupus nephritis, an effective amountof the drug may have the effect in and/or relieving to some extent oneor more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective amount of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective amount” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

“CD20” as used herein refers to the human B-lymphocyte antigen CD20(also known as CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5,and LF5; the sequence is characterized by the SwissProt database entryP11836) is a hydrophobic transmembrane protein with a molecular weightof approximately 35 kD located on pre-B and mature B lymphocytes.(Valentine, M. A., et al., J. Biol. Chem. 264(19) (1989 11282-11287;Tedder, T. F., et al, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 208-12;Stamenkovic, I., et al., J. Exp. Med. 167 (1988) 1975-80; Einfeld, D.A., et al., EMBO J. 7 (1988) 711-7; Tedder, T. F., et al., J. Immunol.142 (1989) 2560-8). The corresponding human gene is Membrane-spanning4-domains, subfamily A, member 1, also known as MS4A1. This gene encodesa member of the membrane-spanning 4A gene family. Members of thisnascent protein family are characterized by common structural featuresand similar intron/exon splice boundaries and display unique expressionpatterns among hematopoietic cells and nonlymphoid tissues. This geneencodes the B-lymphocyte surface molecule which plays a role in thedevelopment and differentiation of B-cells into plasma cells. Thisfamily member is localized to 11q12, among a cluster of family members.Alternative splicing of this gene results in two transcript variantswhich encode the same protein.

The terms “CD20” and “CD20 antigen” are used interchangeably herein, andinclude any variants, isoforms and species homologs of human CD20 whichare naturally expressed by cells or are expressed on cells transfectedwith the CD20 gene. Binding of an antibody of the invention to the CD20antigen mediate the killing of cells expressing CD20 (e.g., a tumorcell) by inactivating CD20. The killing of the cells expressing CD20 mayoccur by one or more of the following mechanisms: Cell death/apoptosisinduction, ADCC and CDC.

Synonyms of CD20, as recognized in the art, include B-lymphocyte antigenCD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5.

The term “anti-CD20 antibody” according to the invention is an antibodythat binds specifically to CD20 antigen. Depending on binding propertiesand biological activities of anti-CD20 antibodies to the CD20 antigen,two types of anti-CD20 antibodies (type I and type II anti-CD20antibodies) can be distinguished according to Cragg, M. S., et al.,Blood 103 (2004) 2738-2743; and Cragg, M. S., et al., Blood 101 (2003)1045-1052, see Table 1 below.

TABLE 1 Properties of type I and type II anti-CD20 antibodies Type Ianti-CD20 antibodies type II anti-CD20 antibodies type I CD20 epitopetype II CD20 epitope Localize CD20 to lipid rafts Do not localize CD20to lipid rafts Increased CDC (if IgG1 isotype) Decreased CDC (if IgG1isotype) ADCC activity (if IgG1 isotype) ADCC activity (if IgG1 isotype)Full binding capacity Reduced binding capacity Homotypic aggregationStronger homotypic aggregation Apoptosis induction upon Strong celldeath induction without cross-linking cross-linking

Examples of type II anti-CD20 antibodies include e.g. humanized B-Ly1antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO2005/044859), 11B8 IgG1 (as disclosed in WO 2004/035607), and AT80 IgG1.Typically type II anti-CD20 antibodies of the IgG1 isotype showcharacteristic CDC properties. Type II anti-CD20 antibodies have adecreased CDC (if IgG1 isotype) compared to type I antibodies of theIgG1 isotype.

Examples of type I anti-CD20 antibodies include e.g. rituximab, HI47IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO 2005/103081), 2F2IgG1 (as disclosed and WO 2004/035607 and WO 2005/103081) and 2H7 IgG1(as disclosed in WO 2004/056312).

The afucosylated anti-CD20 antibodies according to the invention arepreferably type II anti-CD20 antibodies, more preferably afucosylatedhumanized B-Lyl antibodies as described in WO 2005/044859 and WO2007/031875.

The “rituximab” antibody (reference antibody; example of a type Ianti-CD20 antibody) is a genetically engineered chimeric human gamma 1murine constant domain containing monoclonal antibody directed againstthe human CD20 antigen. However this antibody is not glycoengineered andnot afocusylates and thus has an amount of fucose of at least 85%. Thischimeric antibody contains human gamma 1 constant domains and isidentified by the name “C2B8” in U.S. Pat. No. 5,736,137 (Andersen, et.al.) issued on Apr. 17, 1998, assigned to IDEC PharmaceuticalsCorporation. Rituximab is approved for the treatment of patients withrelapsed or refracting low-grade or follicular, CD20 positive, B cellnon-Hodgkin's lymphoma. In vitro mechanism of action studies have shownthat rituximab exhibits human complement-dependent cytotoxicity (CDC)(Reff, M. E., et. al, Blood 83(2) (1994) 435-445). Additionally, itexhibits activity in assays that measure antibody-dependent cellularcytotoxicity (ADCC).

The term “GA101 antibody” as used herein refers to any one of thefollowing antibodies that bind human CD20: (1) an antibody comprising anHVR-H1 comprising the amino acid sequence of SEQ ID NO:1, an HVR-H2comprising the amino acid sequence of SEQ ID NO:2, an HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:3, an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:4, an HVR-L2 comprising the amino acidsequence of SEQ ID NO:5, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO:6; (2) an antibody comprising a VH domaincomprising the amino acid sequence of SEQ ID NO:7 and a VL domaincomprising the amino acid sequence of SEQ ID NO:8, (3) an antibodycomprising an amino acid sequence of SEQ ID NO:9 and an amino acidsequence of SEQ ID NO: 10; (4) an antibody known as obinutuzumab, or (5)an antibody that comprises an amino acid sequence that has at least 95%,96%, 97%, 98% or 99% sequence identity with amino acid sequence of SEQID NO:9 and that comprises an amino acid sequence that has at least 95%,96%, 97%, 98% or 99% sequence identity with an amino acid sequence ofSEQ ID NO:10. In one embodiment, the GA101 antibody is an IgG1 isotypeantibody. In some embodiments, the anti-CD20 antibody is a humanizedB-Ly1 antibody.

The term “humanized B-Ly1 antibody” refers to humanized B-Ly1 antibodyas disclosed in WO 2005/044859 and WO 2007/031875, which were obtainedfrom the murine monoclonal anti-CD20 antibody B-Ly1 (variable region ofthe murine heavy chain (VH): SEQ ID NO: 11; variable region of themurine light chain (VL): SEQ ID NO: 12—see Poppema, S. and Visser, L.,Biotest Bulletin 3 (1987) 131-139) by chimerization with a humanconstant domain from IgG1 and following humanization (see WO 2005/044859and WO 2007/031875). These “humanized B-Ly1 antibodies” are disclosed indetail in WO 2005/044859 and WO 2007/031875.

Variable region of the murine monoclonal anti-CD20 antibody B-Ly1 heavychain (VH) (SEQ ID NO: 11)

Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys  1               5                  10         Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr         15                  20                  Ser Trp Met Asn Trp Val Lys Leu Arg Pro Gly Gln 25                  30                  35      Gly Leu Glu Trp Ile Gly Arg Ile Phe Pro Gly Asp            40                  45    Gly Asp Thr Asp Tyr Asn Gly Lys Phe Lys Gly Lys     50                  55                  60Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala                 65                  70        Tyr Met Gln Leu Thr Ser Leu Thr Ser Val Asp Ser         75                  80                Ala Val Tyr Leu Cys Ala Arg Asn Val Phe Asp Gly 85                  90                  95    Tyr Trp Leu Val Tyr Trp Gly Gln Gly Thr Leu Val             100                 105             Thr Val Ser Ala     110

Variable region of the murine monoclonal anti-CD20 antibody B-Ly1 lightchain (VL) (SEQ ID NO: 12)

Asn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser  1               5                  10     Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly         15                  20      Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly 25                  30                  35    Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn             40                  45     Leu Val Ser Gly Val Pro Asp Arg Phe Ser Ser Ser     50                  55                  60 Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg                 65                  70         Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala         75                  80                Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly 85                  90                  95  Thr Lys Leu Glu Ile Lys Arg            100

In one embodiment, the “humanized B-Ly1 antibody” has variable region ofthe heavy chain (VH) selected from group of SEQ ID NO:7, 8, and 13 to 33(corresponding to, inter alia, B-HH2 to B-HH9 and B-HL8 to B-HL17 of WO2005/044859 and WO 2007/031875). In one specific embodiment, suchvariable domain is selected from the group consisting of SEQ ID NOS:14,15, 7, 19, 25, 27, and 29 (corresponding to B-HH2, BHH-3, B-HH6, B-HH8,B-HL8, B-HL11 and B-HL13 of WO 2005/044859 and WO 2007/031875). In onespecific embodiment, the “humanized B-Ly1 antibody” has variable regionof the light chain (VL) of SEQ ID NO:8 (corresponding to B-KV1 of WO2005/044859 and WO 2007/031875). In one specific embodiment, the“humanized B-Ly1 antibody” has a variable region of the heavy chain (VH)of SEQ ID NO:7 (corresponding to B-HH6 of WO 2005/044859 and WO2007/031875) and a variable region of the light chain (VL) of SEQ IDNO:8 (corresponding to B-KV1 of WO 2005/044859 and WO 2007/031875).Furthermore in one embodiment, the humanized B-Ly1 antibody is an IgG1antibody. According to the invention such afocusylated humanized B-Ly1antibodies are glycoengineered (GE) in the Fc region according to theprocedures described in WO 2005/044859, WO 2004/065540, WO 2007/031875,Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180 and WO 99/154342.In one embodiment, the afucosylated glyco-engineered humanized B-Ly1 isB-HH6-B-KV1 GE. In one embodiment, the anti-CD20 antibody isobinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4,2012, p. 453). As used herein, obinutuzumab is synonymous for GA101 orRO5072759. This replaces all previous versions (e.g. Vol. 25, No. 1,2011, p. 75-′76), and is formerly known as afutuzumab (recommended INN,WHO Drug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2,2008, p. 124). In some embodiments, the humanized B-Ly1 antibody is anantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQID NO:10 or an antigen-binding fragment thereof. In some embodiments,the humanized B-Ly1 antibody comprises a heavy chain variable regioncomprising the three heavy chain CDRs of SEQ ID NO:9 and a light chainvariable region comprising the three light chain CDRs of SEQ ID NO:10.

Heavy chain  (SEQ ID NO: 9)QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA PGQGLEWMGR 50IFPGDGDTDY NGKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARNV 100FDGYWLVYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD 150YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY 200ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK 250DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV 350YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 400DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG 449 Light chain (SEQ ID NO: 10) DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ50 LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQNLELP 100YTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK 150VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE 200VTHQGLSSPV TKSFNRGEC 219

In some embodiments, the humanized B-Ly1 antibody is an afucosylatedglyco-engineered humanized B-Ly1. Such glycoengineered humanized B-Ly1antibodies have an altered pattern of glycosylation in the Fc region,preferably having a reduced level of fucose residues. Preferably theamount of fucose is 60% or less of the total amount of oligosaccharidesat Asn297 (in one embodiment the amount of fucose is between 40% and60%, in another embodiment the amount of fucose is 50% or less, and instill another embodiment the amount of fucose is 30% or less).Furthermore the oligosaccharides of the Fc region are preferablybisected. These glycoengineered humanized B-Ly1 antibodies have anincreased ADCC.

The “ratio of the binding capacities to CD20 on Raji cells (ATCC-No.CCL-86) of an anti-CD20 antibodies compared to rituximab” is determinedby direct immunofluorescence measurement (the mean fluorescenceintensities (MFI) is measured) using said anti-CD20 antibody conjugatedwith Cy5 and rituximab conjugated with Cy5 in a FACSArray (BectonDickinson) with Raji cells (ATCC-No. CCL-86), as described in ExampleNo. 2, and calculated as follows:

${{Ratio}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {binding}\mspace{14mu} {capacities}\mspace{14mu} {to}\mspace{14mu} {CD}\; 20\mspace{14mu} {on}\mspace{14mu} {Raji}\mspace{14mu} {cells}\mspace{14mu} \left( {{ATCC}\text{-}{{No}.\; {CCL}}\text{-}86} \right)} = \mspace{79mu} {\frac{{MFI}\left( {{Cy}\; 5\text{-}{anti}\text{-}{CD}\; 20\mspace{14mu} {antibody}} \right)}{{MFI}\left( {{Cy}\; 5\text{-}{rituximab}} \right)} \times \frac{{Cy}\; 5\text{-}{labeling}\mspace{14mu} {{ratio}\left( {{Cy}\; 5\text{-}{rituximab}} \right)}}{{Cy}\; 5\text{-}{labeling}\mspace{14mu} {{ratio}\left( {{Cy}\; 5\text{-}{anti}\text{-}{CD}\; 20\mspace{14mu} {antibody}} \right)}}}$

MFI is the mean fluorescent intensity. The “Cy5-labeling ratio” as usedherein means the number of Cy5-label molecules per molecule antibody.

Typically said type II anti-CD20 antibody has a ratio of the bindingcapacities to CD20 on Raji cells (ATCC-No. CCL-86) of said secondanti-CD20 antibody compared to rituximab of 0.3 to 0.6, and in oneembodiment, 0.35 to 0.55, and in yet another embodiment, 0.4 to 0.5.

In one embodiment said type II anti-CD20 antibody, e.g., a GA101antibody, has increased antibody dependent cellular cytotoxicity (ADCC).

By “antibody having increased antibody dependent cellular cytotoxicity(ADCC)”, it is meant an antibody, as that term is defined herein, havingincreased ADCC as determined by any suitable method known to those ofordinary skill in the art. One accepted in vitro ADCC assay is asfollows:

-   -   1) the assay uses target cells that are known to express the        target antigen recognized by the antigen-binding region of the        antibody;    -   2) the assay uses human peripheral blood mononuclear cells        (PBMCs), isolated from blood of a randomly chosen healthy donor,        as effector cells;    -   3) the assay is carried out according to following protocol:        -   i) the PBMCs are isolated using standard density            centrifugation procedures and are suspended at 5×10⁶            cells/ml in RPMI cell culture medium;        -   ii) the target cells are grown by standard tissue culture            methods, harvested from the exponential growth phase with a            viability higher than 90%, washed in RPMI cell culture            medium, labeled with 100 micro-Curies of ⁵¹Cr, washed twice            with cell culture medium, and resuspended in cell culture            medium at a density of 10⁵ cells/ml;        -   iii) 100 microliters of the final target cell suspension            above are transferred to each well of a 96-well microtiter            plate;        -   iv) the antibody is serially-diluted from 4000 ng/ml to 0.04            ng/ml in cell culture medium and 50 microliters of the            resulting antibody solutions are added to the target cells            in the 96-well microtiter plate, testing in triplicate            various antibody concentrations covering the whole            concentration range above;        -   v) for the maximum release (MR) controls, 3 additional wells            in the plate containing the labeled target cells, receive 50            microliters of a 2% (VN) aqueous solution of non-ionic            detergent (Nonidet, Sigma, St. Louis), instead of the            antibody solution (point iv above);        -   vi) for the spontaneous release (SR) controls, 3 additional            wells in the plate containing the labeled target cells,            receive 50 microliters of RPMI cell culture medium instead            of the antibody solution (point iv above);        -   vii) the 96-well microtiter plate is then centrifuged at            50×g for 1 minute and incubated for 1 hour at 4° C.;        -   viii) 50 microliters of the PBMC suspension (point i above)            are added to each well to yield an effector:target cell            ratio of 25:1 and the plates are placed in an incubator            under 5% CO2 atmosphere at 37° C. for 4 hours;        -   ix) the cell-free supernatant from each well is harvested            and the experimentally released radioactivity (ER) is            quantified using a gamma counter;        -   x) the percentage of specific lysis is calculated for each            antibody concentration according to the formula            (ER−MR)/(MR−SR)×100, where ER is the average radioactivity            quantified (see point ix above) for that antibody            concentration, MR is the average radioactivity quantified            (see point ix above) for the MR controls (see point V            above), and SR is the average radioactivity quantified (see            point ix above) for the SR controls (see point vi above);    -   4) “increased ADCC” is defined as either an increase in the        maximum percentage of specific lysis observed within the        antibody concentration range tested above, and/or a reduction in        the concentration of antibody required to achieve one half of        the maximum percentage of specific lysis observed within the        antibody concentration range tested above. In one embodiment,        the increase in ADCC is relative to the ADCC, measured with the        above assay, mediated by the same antibody, produced by the same        type of host cells, using the same standard production,        purification, formulation and storage methods, which are known        to those skilled in the art, except that the comparator antibody        (lacking increased ADCC) has not been produced by host cells        engineered to overexpress GnTIII and/or engineered to have        reduced expression from the fucosyltransferase 8 (FUT8) gene        (e.g., including, engineered for FUT8 knock out).

Said “increased ADCC” can be obtained by, for example, mutating and/orglycoengineering of said antibodies. In one embodiment, the antibody isglycoengineered to have a biantennary oligosaccharide attached to the Fcregion of the antibody that is bisected by GlcNAc, e.g., in WO2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana etal.); US 2005/0123546 (Umana et al.), Umana, P., et al., NatureBiotechnol. 17 (1999) 176-180). In another embodiment, the antibody isglycoengineered to lack fucose on the carbohydrate attached to the Fcregion by expressing the antibody in a host cell that is deficient inprotein fucosylation (e.g., Lec13 CHO cells or cells having analpha-1,6-fucosyltransferase gene (FUT8) deleted or the FUT geneexpression knocked down (see, e.g., Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng.,94(4):680-688 (2006); and WO2003/085107). In yet another embodiment, theantibody sequence has been engineered in its Fc region to enhance ADCC(e.g., in one embodiment, such engineered antibody variant comprises anFc region with one or more amino acid substitutions at positions 298,333, and/or 334 of the Fc region (EU numbering of residues)).

The term “complement-dependent cytotoxicity (CDC)” refers to lysis ofhuman tumor target cells by the antibody according to the invention inthe presence of complement. CDC can be measured by the treatment of apreparation of CD20 expressing cells with an anti-CD20 antibodyaccording to the invention in the presence of complement. CDC is foundif the antibody induces at a concentration of 100 nM the lysis (celldeath) of 20% or more of the tumor cells after 4 hours. In oneembodiment, the assay is performed with ⁵¹Cr or Eu labeled tumor cellsand measurement of released ⁵¹Cr or Eu. Controls include the incubationof the tumor target cells with complement but without the antibody.

The term “expression of the CD20” antigen is intended to indicate asignificant level of expression of the CD20 antigen in a cell, e.g., aT- or B-Cell. In one embodiment, patients to be treated according to themethods of this invention express significant levels of CD20 on aB-cell. CD20 expression on a B-cell can be determined by standard assaysknown in the art. e.g., CD20 antigen expression is measured usingimmunohistochemical (IHC) detection, FACS or via PCR-based detection ofthe corresponding mRNA.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

III. METHODS

In one aspect, provided herein are methods for treating or delayingprogression of lupus nephritis in an individual that has lupus byadministering an effective amount of a type II anti-CD20 antibody. Insome embodiments, the individual has or is at risk for developing lupusnephritis. In some embodiments, the lupus nephritis is class III orclass IV lupus nephritis. In some embodiments, the methods includeadministering to the individual at least a first antibody exposure to atype II anti-CD20 antibody and a second antibody exposure to the type IIanti-CD20 antibody, the second antibody exposure not being provideduntil from about 18 weeks to about 26 weeks after the first antibodyexposure; wherein the first antibody exposure comprises one or two dosesof the type II anti-CD20 antibody, the first antibody exposurecomprising a total exposure of between about 1800 mg and about 2200 mgof the type II anti-CD20 antibody; and wherein the second antibodyexposure comprises one or two doses of the type II anti-CD20 antibody,the second antibody exposure comprising a total exposure of betweenabout 1800 mg and about 2200 mg of the type II anti-CD20 antibody. Asdescribed below, in some embodiments, the antibody comprises a heavychain comprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequence of SEQID NO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chaincomprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ IDNO:5, and HVR-L3 sequence of SEQ ID NO:6. In some embodiments, theantibody comprises a VH domain comprising the amino acid sequence of SEQID NO:7 and a VL domain comprising the amino acid sequence of SEQ IDNO:8. In some embodiments, the antibody comprises an amino acid sequenceof SEQ ID NO:9 and an amino acid sequence of SEQ ID NO:10. In someembodiments, the antibody comprises an antibody that comprises an aminoacid sequence that has at least 95%, 96%, 97%, 98% or 99% sequenceidentity with amino acid sequence of SEQ ID NO:9 and that comprises anamino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequenceidentity with an amino acid sequence of SEQ ID NO:10.

Anti-CD20 Antibodies

Certain aspects of the present disclosure relate to anti-CD20antibodies, e.g., for use in methods for treating or preventingprogression of lupus nephritis. In some embodiments, the anti-CD20antibody is a type II antibody. In some embodiments, the anti-CD20antibody is human or humanized. In some embodiments, the anti-CD20antibody is afucosylated. In some embodiments, the anti-CD20 antibody isa GA101 antibody.

Examples of type II anti-CD20 antibodies include e.g. humanized B-Ly1antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO2005/044859), 11B8 IgG1 (as disclosed in WO 2004/035607), and AT80 IgG1.Typically type II anti-CD20 antibodies of the IgG1 isotype showcharacteristic CDC properties. Type II anti-CD20 antibodies have adecreased CDC (if IgG1 isotype) compared to type I antibodies of theIgG1 isotype.

Examples of type I anti-CD20 antibodies include e.g. rituximab, HI47IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO 2005/103081), 2F2IgG1 (as disclosed and WO 2004/035607 and WO 2005/103081) and 2H7 IgG1(as disclosed in WO 2004/056312).

In some embodiments, the anti-CD20 antibody is a GA101 antibodydescribed herein. In some embodiments, the anti-CD20 is any one of thefollowing antibodies that bind human CD20: (1) an antibody comprising anHVR-H1 comprising the amino acid sequence of GYAFSY (SEQ ID NO:1), anHVR-H2 comprising the amino acid sequence of FPGDGDTD (SEQ ID NO:2), anHVR-H3 comprising the amino acid sequence of NVFDGYWLVY (SEQ ID NO:3),an HVR-L1 comprising the amino acid sequence of RSSKSLLHSNGITYLY (SEQ IDNO:4), an HVR-L2 comprising the amino acid sequence of QMSNLVS (SEQ IDNO:5), and an HVR-L3 comprising the amino acid sequence of AQNLELPYT(SEQ ID NO:6); (2) an antibody comprising a VH domain comprising theamino acid sequence of SEQ ID NO:7 and a VL domain comprising the aminoacid sequence of SEQ ID NO:8, (3) an antibody comprising an amino acidsequence of SEQ ID NO:9 and an amino acid sequence of SEQ ID NO:10; (4)an antibody known as obinutuzumab, or (5) an antibody that comprises anamino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequenceidentity with amino acid sequence of SEQ ID NO:9 and that comprises anamino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequenceidentity with an amino acid sequence of SEQ ID NO:10. In one embodiment,the GA101 antibody is an IgG1 isotype antibody. In some embodiments, theanti-CD20 antibody comprises an HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2,and HVR-L3 of any of the antibodies described herein, e.g., 3 HVRs fromSEQ ID NO:7 and 3 HVRs from SEQ ID NO:8, 3 HVRs from SEQ ID NO:9 and 3HVRs from SEQ ID NO:10, or any HVRs of the amino acid sequences providedin Table 2.

In some embodiments, the anti-CD20 antibody comprises a heavy chainvariable region (VH) comprising the amino acid sequence of SEQ ID NO:7,and a light chain variable region (VL) comprising the amino acidsequence of SEQ ID NO:8.

(SEQ ID NO: 7) QVQLVQSGAEVKKPGSSVKVSCKAS GYAFSY SWINWVRQAPGQGLEWMGR IFPGDGDTD YNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR NV FDGYWLVYWGQGTLVTVSS  (SEQ ID NO: 8) DIVMTQTPLSLPVTPGEPASISC RSSKSLLHSNGITYLYWYLQKPGQSPQ LLIY Q MSNLVS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC AQNLELP YTFGGGTKVEIKRTV.

In some embodiments, the anti-CD20 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:9, and a light chaincomprising the amino acid sequence of SEQ ID NO:10.

(SEQ ID NO: 9) QVQLVQSGAEVKKPGSSVKVSCKAS

SWINWVRQAPGQGLEW MGRI

YNGKFKGRVTITADKSTSTAYMELSSLRSEDTAV YYCAR

WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG(SEQ ID NO: 10) DIVMTQTPLSLPVTPGEPASISC

WYLQKPGQ SPQLLIY

GVPDRFSGSGSGTDFILKISRVEAEDVGVYYC

EGGGIKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In some embodiments, the anti-CD20 antibody is a humanized B-Ly1antibody. In some embodiments, the humanized B-Ly1 antibody comprises aheavy chain variable region comprising the three heavy chain CDRs of SEQID NO:9 and a light chain variable region comprising the three lightchain CDRs of SEQ ID NO:10. In some embodiments, the humanized B-Ly1antibody comprises a heavy chain comprising the sequence of SEQ ID NO:9and a light chain comprising the sequence of SEQ ID NO:10.

In some embodiments, the anti-CD20 antibody comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toa polypeptide sequence listed in Table 2 below.

TABLE 2  Polypeptide sequences. SEQ ID CONSTRUCT POLYPEPTIDE SEQUENCE NOB-HH1 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSYSWM 13SWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HH2 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM 14NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HH3 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM 15NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYLCARNVFDGYWLVYWGQGTLVTVSS B-HH4 QVQLVQSGAEVKKPGASVKVSCKVSGYAFSYSWM 16NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HH5 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM 17SWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HH6 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWIN 7WVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HH7 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWIS 18WVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HH8 QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWM 19NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HH9 QVQLVQSGAEVKKPGASVKVSCKASGYTFSYSWM 20NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWM 21HWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGR VTMTRDTSTSTVYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL2 EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWMH 22WVQQAPGKGLEWMGRIFPGDGDTDYAEKFQGRVT ITADTSTDTAYMELSSLRSEDTAVYYCATNVFDGYWLVYWGQGTLVTVSS B-HL3 EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWMN 23WVQQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT ITADTSTDTAYMELSSLRSEDTAVYYCATNVFDGYWLVYWGQGTLVTVSS B-HL4 QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYSWM 24SWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL8 EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMN 25WVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL10 EVQLVESGGGLVKPGGSLRLSCAASGFAFSYSWMN 26WVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL11 QVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMN 27WVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL12 EVQLVESGAGLVKPGGSLRLSCAASGFTFSYSWMN 28WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL13 EVQLVESGGGVVKPGGSLRLSCAASGFTFSYSWMN 29WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL14 EVQLVESGGGLKKPGGSLRLSCAASGFTFSYSWMN 30WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL15 EVQLVESGGGLVKPGSSLRLSCAASGFTFSYSWMN 31WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL16 EVQLVESGGGLVKPGGSLRVSCAASGFTFSYSWMN 32WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS B-HL17 EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMN 33WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS VH Signal MDWTWRILFLVAAATGAHS 34 Sequence B-KV1DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYL 8YWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTV VL Signal MDMRVPAQLLGLLLLWFPGARC 43 Sequence

In some embodiments, the anti-CD20 antibody (e.g., a type II anti-CD20antibody) is an afucosylated glyco-engineered antibody. Suchglycoengineered antibodies have an altered pattern of glycosylation inthe Fc region, preferably having a reduced level of fucose residues.Preferably the amount of fucose is 60% or less of the total amount ofoligosaccharides at Asn297 (in one embodiment the amount of fucose isbetween 40% and 60%, in another embodiment the amount of fucose is 50%or less, and in still another embodiment the amount of fucose is 30% orless). Furthermore the oligosaccharides of the Fc region are preferablybisected. These glycoengineered humanized anti-CD20 (e.g., B-Ly1)antibodies have an increased ADCC.

The oligosaccharide component can significantly affect propertiesrelevant to the efficacy of a therapeutic glycoprotein, includingphysical stability, resistance to protease attack, interactions with theimmune system, pharmacokinetics, and specific biological activity. Suchproperties may depend not only on the presence or absence, but also onthe specific structures, of oligosaccharides. Some generalizationsbetween oligosaccharide structure and glycoprotein function can be made.For example, certain oligosaccharide structures mediate rapid clearanceof the glycoprotein from the bloodstream through interactions withspecific carbohydrate binding proteins, while others can be bound byantibodies and trigger undesired immune reactions. (Jenkins, N., et al.,Nature Biotechnol. 14 (1996) 975-81).

Mammalian cells are the preferred hosts for production of therapeuticglycoproteins, due to their capability to glycosylate proteins in themost compatible form for human application. (Cumming, D. A., et al.,Glycobiology 1 (1991) 115-30; Jenkins, N., et al., Nature Biotechnol. 14(1996) 975-81). Bacteria very rarely glycosylate proteins, and likeother types of common hosts, such as yeasts, filamentous fungi, insectand plant cells, yield glycosylation patterns associated with rapidclearance from the blood stream, undesirable immune interactions, and insome specific cases, reduced biological activity. Among mammalian cells,Chinese hamster ovary (CHO) cells have been most commonly used duringthe last two decades. In addition to giving suitable glycosylationpatterns, these cells allow consistent generation of genetically stable,highly productive clonal cell lines. They can be cultured to highdensities in simple bioreactors using serum free media, and permit thedevelopment of safe and reproducible bioprocesses. Other commonly usedanimal cells include baby hamster kidney (BHK) cells, NSO- andSP2/0-mouse myeloma cells. More recently, production from transgenicanimals has also been tested. (Jenkins, N., et al., Nature Biotechnol.14 (1996) 975-981).

All antibodies contain carbohydrate structures at conserved positions inthe heavy chain constant regions, with each isotype possessing adistinct array of N-linked carbohydrate structures, which variablyaffect protein assembly, secretion or functional activity. (Wright, A.,and Morrison, S. L., Trends Biotech. 15 (1997) 26-32). The structure ofthe attached N-linked carbohydrate varies considerably, depending on thedegree of processing, and can include high-mannose, multiply-branched aswell as biantennary complex oligosaccharides. (Wright, A., and Morrison,S. L., Trends Biotech. 15 (1997) 26-32). Typically, there isheterogeneous processing of the core oligosaccharide structures attachedat a particular glycosylation site such that even monoclonal antibodiesexist as multiple glycoforms. Likewise, it has been shown that majordifferences in antibody glycosylation occur between cell lines, and evenminor differences are seen for a given cell line grown under differentculture conditions. (Lifely, M. R., et al., Glycobiology 5(8) (1995)813-22).

One way to obtain large increases in potency, while maintaining a simpleproduction process and potentially avoiding significant, undesirableside effects, is to enhance the natural, cell-mediated effectorfunctions of monoclonal antibodies by engineering their oligosaccharidecomponent as described in Umana, P., et al., Nature Biotechnol. 17(1999) 176-180 and U.S. Pat. No. 6,602,684. IgG1 type antibodies, themost commonly used antibodies in cancer immunotherapy, are glycoproteinsthat have a conserved N-linked glycosylation site at Asn297 in each CH2domain. The two complex biantennary oligosaccharides attached to Asn297are buried between the CH2 domains, forming extensive contacts with thepolypeptide backbone, and their presence is essential for the antibodyto mediate effector functions such as antibody dependent cellularcytotoxicity (ADCC) (Lifely, M. R., et al., Glycobiology 5 (1995)813-822; Jefferis, R., et al., Immunol. Rev. 163 (1998) 59-76; Wright,A., and Morrison, S. L., Trends Biotechnol. 15 (1997) 26-32).

It was previously shown that overexpression in Chinese hamster ovary(CHO) cells of β(1,4)-N-acetylglucosaminyltransferase I11 (“GnTII17y), aglycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofan antineuroblastoma chimeric monoclonal antibody (chCE7) produced bythe engineered CHO cells. (See Umana, P., et al., Nature Biotechnol. 17(1999) 176-180; and WO 99/154342, the entire contents of which arehereby incorporated by reference). The antibody chCE7 belongs to a largeclass of unconjugated monoclonal antibodies which have high tumoraffinity and specificity, but have too little potency to be clinicallyuseful when produced in standard industrial cell lines lacking theGnTIII enzyme (Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180).That study was the first to show that large increases of ADCC activitycould be obtained by engineering the antibody producing cells to expressGnTIII, which also led to an increase in the proportion of constantregion (Fc)-associated, bisected oligosaccharides, including bisected,non-fucosylated oligosaccharides, above the levels found innaturally-occurring antibodies.

In some embodiments, the anti-CD20 antibody (e.g., a type II anti-CD20antibody) comprises a human Fc region (e.g., a human IgG1 Fc region). Insome embodiments, the Fc region comprises an N-linked oligosaccharidethat has been modified. In some embodiments, the N-linkedoligosaccharides of the Fc region have reduced fucose residues ascompared to an antibody with non-modified N-linked oligosaccharides. Insome embodiments, the bisected oligosaccharide is a bisected complexoligosaccharide. In some embodiments, the N-linked oligosaccharides havebeen modified to have increased bisected, nonfucosylatedoligosaccharides. In some embodiments, the bisected, nonfucosylatedoligosaccharides are the hybrid type. In some embodiments, the bisected,nonfucosylated oligosaccharides are the complex type. For more detaileddescription, see, e.g., WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.); and U.S.Pat. No. 8,883,980 (Umana et al.).

In some embodiments, the anti-CD20 antibody (e.g., a type II anti-CD20antibody) is a multispecific antibody or a bispecific antibody.

Antibody Preparation

An antibody according to any of the above embodiments (e.g., a type IIanti-CD20 antibody of the present disclosure) may incorporate any of thefeatures, singly or in combination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or≦0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸ M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881(1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CM5 chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 106 M-1 s-1 by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for CD20 and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of CD20. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express CD20. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to CD20 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table A under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table A under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE A Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

In certain embodiments, the Fc variants described herein furthercomprise one or more amino acid modifications for attenuating effectorfunction (such as CDC and/or ADCC). In exemplary embodiments, themodification to attenuate effector function is a modification that doesnot alter the glycosylation pattern of the Fc region. In certainembodiments, the modification to attenuate effector function reduces oreliminates binding to human effector cells, binding to one or more Fcreceptors, and/or binding to cells expressing an Fc receptor. In anexemplary embodiment, the Fc variants described herein comprise thefollowing modifications: L234A, L235A and P329G in the Fc region ofhuman IgG1, that result in attenuated effector function. SubstitutionsL234A, L235A, and P329G (the L234A/L235A/P329G triple variant isreferred to as LALAPG) have previously been shown to reduce binding toFc receptors and complement (see e.g., US Publication No. 2012/0251531).

In various embodiments, Fc variants having reduced effector functionrefer to Fc variants that reduce effector function (e.g., CDC, ADCC,and/or binding to FcR, etc. activities) by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more as compared to theeffector function achieved by a wild-type Fc region (e.g., an Fc regionnot having a mutation to reduce effector function, although it may haveother mutations). In certain embodiments, Fc variants having reducedeffector function refer to Fc variants that eliminate all detectableeffector function as compared to a wild-type Fc region. Assays formeasuring effector function are known in the art and described below.

In vitro and/or in vivo cytotoxicity assays can be conducted to confirmthe reduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity). Theprimary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII FcR expression onhematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev.Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays toassess ADCC activity of a molecule of interest is described in U.S. Pat.No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al.,J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactiveassays methods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).C1q binding assays may also be carried out to confirm that the antibodyis unable to bind C1q and hence lacks CDC activity. See, e.g., C1q andC3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assesscomplement activation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

A. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-CD20 antibody described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-CD20 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-CD20 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.,2003), pp. 245-254, describing expression of antibody fragments in E.coli.) After expression, the antibody may be isolated from the bacterialcell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

B. Assays

Anti-CD20 antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Binding assays and other assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc. CD20 binding may be determined using methods known in the art andexemplary methods are disclosed herein. In one embodiment, binding ismeasured using radioimmunoassay. An exemplary radioimmunoassay isprovided below. CD20 antibody is iodinated, and competition reactionmixtures are prepared containing a fixed concentration of iodinatedantibody and decreasing concentrations of serially diluted, unlabeledCD20 antibody. Cells expressing CD20 (e.g., BT474 cells stablytransfected with human CD20) are added to the reaction mixture.Following an incubation, cells are washed to separate the free iodinatedCD20 antibody from the CD20 antibody bound to the cells. Level of boundiodinated CD20 antibody is determined, e.g., by counting radioactivityassociated with cells, and binding affinity determined using standardmethods. In another embodiment, ability of CD20 antibody to bind tosurface-expressed CD20 (e.g., on B cell subsets) is assessed using flowcytometry. Peripheral white blood cells are obtained (e.g., from human,cynomolgus monkey, rat or mouse) and cells are blocked with serum.Labeled CD20 antibody is added in serial dilutions, and T cells are alsostained to identify T cell subsets (using methods known in the art).Following incubation of the samples and washing, the cells are sortedusing flow cytometer, and data analyzed using methods well known in theart. In another embodiment, CD20 binding may be analyzed using surfaceplasmon resonance. An exemplary surface plasmon resonance method isexemplified in the Examples.

In another aspect, competition assays may be used to identify anantibody that competes with any of the anti-CD20 antibodies disclosedherein for binding to CD20. In certain embodiments, such a competingantibody binds to the same epitope (e.g., a linear or a conformationalepitope) that is bound by any of the anti-CD20 antibodies disclosedherein. Detailed exemplary methods for mapping an epitope to which anantibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.).

In an exemplary competition assay, immobilized CD20 is incubated in asolution comprising a first labeled antibody that binds to CD20 (e.g.,rituximab, a GA101 antibody, etc.) and a second unlabeled antibody thatis being tested for its ability to compete with the first antibody forbinding to CD20. The second antibody may be present in a hybridomasupernatant. As a control, immobilized CD20 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to CD20, excess unbound antibody is removed, and theamount of label associated with immobilized CD20 is measured. If theamount of label associated with immobilized CD20 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to CD20. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity assays

Anti-CD20 antibodies of the present disclosure (e.g., a type IIantibody) may be identified and/or characterized by one or more activityassays known in the art. For example, a complement-dependentcytotoxicity (CDC) and/or antibody-dependent cellular cytotoxicity(ADCC) may be used, as described herein.

It is understood that any of the above assays may be carried out usingan immunoconjugate of the invention in place of or in addition to ananti-CD20 antibody.

It is understood that any of the above assays may be carried out usinganti-CD20 antibody and an additional therapeutic agent.

C. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CD20antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

Methods for Treating or Delaying Progression of Lupus Nephritis

Certain aspects of the present disclosure relate to methods for treatingor delaying progression of lupus nephritis (LN) in an individual thathas lupus. In some embodiments, the individual or patient is a human.

LN is known in the art as a manifestation of lupus (e.g., systemic lupuserythematosus, drug-induced lupus, neonatal lupus, or discoid lupus) inthe kidney(s). The most common type of lupus that manifests in thekidneys is systemic lupus erythematosus (SLE). It is thought that 25-50%of SLE patients have abnormalities in the urine and/or renal functionearly in the course of their disease, with up to 60% of adults and 80%of children eventually developing LN (for more details, see Cameron, J.S. (1999) J. Am. Soc. Nephrol. 10:413-424). LN is thought to account forat least 50% of the morbidity and mortality associated with SLE.

In addition, renal manifestations have also been noted in other types oflupus, such as discoid (Roujeau, J. C. et al. (1984) Acta Derm.Venereol. 64:160-163) and drug-induced lupus (Smith, P. R. et al. (1999)Rheumatology (Oxford) 38:1017-1018). In some embodiments, the individualhas SLE, discoid lupus, or drug-induced lupus.

Diagnosis of SLE may be according to current American College ofRheumatology (ACR) criteria. Active disease may be defined by oneBritish Isles Lupus Activity Group's (BILAG) “A” criteria or two BILAG“B” criteria; SLE Disease Activity Index (SLEDAI); or systemic lupuserythematosus (SLE) responder index (SRI) as noted in the Examples belowand descrived in Furie et al., Arthritis Rheum. 61(9):1143-51 (2009).Some signs, symptoms, or other indicators used to diagnose SLE adaptedfrom: Tan et al. “The Revised Criteria for the Classification of SLE”Arth Rheum 25 (1982) may be malar rash such as rash over the cheeks,discoid rash, or red raised patches, photosensitivity such as reactionto sunlight, resulting in the development of or increase in skin rash,oral ulcers such as ulcers in the nose or mouth, usually painless,arthritis, such as non-erosive arthritis involving two or moreperipheral joints (arthritis in which the bones around the joints do notbecome destroyed), serositis, pleuritis or pericarditis, renal disordersuch as excessive protein in the urine (greater than 0.5 gm/day or 3+ ontest sticks) and/or cellular casts (abnormal elements derived from theurine and/or white cells and/or kidney tubule cells), neurologic signs,symptoms, or other indicators, seizures (convulsions), and/or psychosisin the absence of drugs or metabolic disturbances that are known tocause such effects, and hematologic signs, symptoms, or other indicatorssuch as hemolytic anemia or leukopenia (white blood count below 4,000cells per cubic millimeter) or lymphopenia (less than 1,500 lymphocytesper cubic millimeter) or thrombocytopenia (less than 100,000 plateletsper cubic millimeter). The leukopenia and lymphopenia must be detectedon two or more occasions. The thrombocytopenia must be detected in theabsence of drugs known to induce it. The invention is not limited tothese signs, symptoms, or other indicators of lupus.

The presence of autoantibodies may be tested as an indication for lupus.Autoantibodies may include without limitation anti-dsDNA antibodies,anti-complement antibodies, and antinuclear antibodies (e.g., an ENApanel). ENA refers to Extractable Nuclear Antigens, i.e., a group ofnuclear antigens including, e.g., RNP, Ro/SS-A, La/SS-B, Sm, SCL-70,Jo-1, as described in McNeilage et al., J., Clin. Lab. Immunol. 15:1-17(1984); Whittingham, Ann. Acad. Med. 17(2):195-200 (1988); Wallace andHahn, DUBOIS' LUPUS ERYTHEMATOSUS, 7^(TH) ED. LIPPINCOTT (2007); Tang etal., Medicine 89(1): 62-67 (2010). Antibodies to ENA have beencorrelated to lupus. McNeilage et al., 1984; Whittingham 1988; Ashersonet al., Medicine 68(6): 366-374 (1989); and Tang et al., 2010. Reducedcomplement activity may also be associated with lupus, e.g., as measuredby C3 levels, C4 levels, and/or a CH50 assay.

As described above in reference to SLE, it is known in the art that LNoften manifests progressively in patients with lupus (e.g., systemiclupus erythematosus, drug-induced lupus, neonatal lupus, or discoidlupus). That is to say, a patient may be diagnosed with lupus without aclinical or pathological manifestation of one or more LN symptoms.Nonetheless, the patient may still be considered to be at risk fordeveloping LN due to the high frequency of lupus patients thateventually develop LN. Therefore, in some embodiments, the methods ofthe present disclosure may find use in delaying progression of LN, orpreventing LN, in a patient with lupus. In some embodiments, the methodsof the present disclosure may find use in postponing or preventing theonset of LN in a patient with lupus (e.g., a form of lupus that lacks amanifestation in the kidney(s)).

LN pathology may be classified according to the International Society ofNephrology/Renal Pathology Society (ISN/RPS) 2003 classification system,as shown in the table below (see Markowitz G S, D'Agati V D (2007)Kidney Int 71:491-495 and Weening, J J (2004) Kidney Int 65:521-530 forfurther descriptions and definitions of terms).

TABLE 3 ISN/RPS 2003 Classification of Lupus Nephritis. Class Minimalmesangial LN I (Normal glomeruli by light microscopy, but mesangialimmune deposits by immunofluorescence) Class Mesangial proliferative LNII (Purely mesangial hypercellularity of any degree or mesangial matrixexpansion by light microscopy, with mesangial immune deposits. A fewisolated subepithelial or subendothelial deposits may be visible byimmunofluorescence or electron microscopy, but not by light microscopy)Class Focal LN III (Active or inactive focal, segmental or globalendo-or extracapillary glomerulonephritis involving <50% of allglomeruli, typically with focal subendothelial immune deposits, with orwithout mesangial alterations) III (A): active lesions (focalproliferative LN) III (A/C): active and chronic lesions (focalproliferative and sclerosing LN) III (C): chronic inactive lesions withglomerular scars (focal sclerosing LN) Class Diffuse LN IV (Active orinactive diffuse, segmental or global endo-or extracapillaryglomerulonephritis involving ≧50% of all glomeruli, typically withdiffuse subendothelial immune deposits, with or without mesangialalterations. This class is divided into diffuse segmental (IV-S) LN when≧50% of the involved glomeruli have segmental lesions, and diffuseglobal (IV-G) LN when ≧50% of the involved glomeruli have globallesions. Segmental is defined as a glomerular lesion that involves lessthan half of the glomerular tuft. This class includes cases with diffusewire loop deposits but with little or no glomerular proliferation.) IV-S(A): active lesions (diffuse segmental proliferative LN) IV-G (A):active lesions (diffuse global proliferative LN) IV-S (A/C): active andchronic lesions (diffuse segmental proliferative and sclerosing LN) IV-G(A/C): active and chronic lesions (diffuse global proliferative andsclerosing LN) IV-S (C): chronic inactive lesions with scars (diffusesegmental sclerosing LN) IV-G (C): chronic inactive lesions with scars(diffuse global sclerosing LN) Class Membranous LN V (Global orsegmental subepithelial immune deposits or their morphologic sequelae bylight microscopy and by immuno- fluorescence or electron microscopy,with or without mesangial alterations.) Class Advanced sclerotic LN VI90% of glomeruli globally sclerosed without residual activity) LN =lupus nephritis; A = active; C = chronic; G = global; S = segmental.Note: Class V may occur in combination with Class III or IV, in whichcase both will be diagnosed. Class V LN may show advanced sclerosis.

In some embodiments, the patient has class III or class IV LN. In someembodiments, the patient has class III LN. For example, in someembodiments, the patient has class III(A) or class III(A/C) LN. In someembodiments, the patient has class IV LN. For example, in someembodiments, the patient has class IV-S(A), IV-G(A), IV-S(A/C), orIV-G(A/C) LN. As shown in Table 3 above, class V LN may also occurconcomitantly with class III or class IV LN. In some embodiments, themethods of the present disclosure are used to treat a patient with classIII or class IV LN and concomitant class V LN.

As discussed above, a high frequency of patients with lupus (e.g., SLE)eventually develop LN. In some embodiments, the patient is at risk fordeveloping LN. In some embodiments, the patient is at risk fordeveloping class III or class IV LN. In some embodiments, the patient isat risk for developing class III or class IV LN with concomitant class VLN.

In some embodiments, the patient does not have class III(C) LN (e.g., asdescribed in Table 3 above). In some embodiments, the patient does nothave class IV(C) LN, such as class IV-S(C) or IV-G(C) LN (e.g., asdescribed in Table 3 above).

Several lab tests known in the art may be used to diagnose and/ormonitor the presence, progression, and/or response to treatment in lupusnephritis. In some embodiments, serum creatinine may be measured. Insome embodiments, the normal range for serum creatinine may be fromabout 0.6 to about 1.3 mg/dL, with some variation seen by age, betweenmen and women, and from lab to lab. In some embodiments, the presence ofurinary sediment and/or casts may be measured, e.g., by microscopicexamination of urine. For example, the number of red blood cells in aurine sample may be assayed by microscopic examination. In someembodiments, a normal value for urinary sediment may be about 4 redblood cells (RBC) or less per high power field (HPF). Urinary casts mayinclude without limitation red blood cell casts, white blood cell casts,renal tubular epithelial cell casts, waxy casts, hyaline casts, granularcasts, and fatty casts. In some embodiments, a urinary protein tocreatinine ratio (UPCR) may be measured. The presence of protein in theurine (proteinuria) may also be assayed by tests including withoutlimitation a urine albumin to creatinine ratio (UACR) and dipstickurinalysis. Other tests and/or measures that may be useful for examiningrenal function include without limitation a renal panel, creatinineclearance, sodium, potassium, chloride, bicarbonate, phosphorus,calcium, albumin, blood urea nitrogen (BUN), creatinine, glucose,estimated glomerular filtration rate (eGFR), BUN/creatinine ratio, andanion gap, and may include a measurement of the above parameters in theblood and/or urine, where appropriate. For more detailed description,see, e.g., the American College of Rheumatology Guidelines forScreening, Case Definition, Treatment and Management of Lupus Nephritis(Hahn, B. et al. (2012) Arthritis Care Res. 64:797-808).

In some embodiments, the methods of the present disclosure includeadministering to the individual at least a first antibody exposure to atype II anti-CD20 antibody of the present disclosure and a secondantibody exposure to the type II anti-CD20 antibody. Any of the type IIanti-CD20 antibodies described herein may be used, e.g., a GA101antibody such as obinutuzumab. In some embodiments, the second antibodyexposure is not provided until from about 18 weeks to about 26 weeksafter the first antibody exposure. In some embodiments, the secondantibody exposure is not provided until about 18 weeks after the firstantibody exposure, about 19 weeks after the first antibody exposure,about 20 weeks after the first antibody exposure, about 21 weeks afterthe first antibody exposure, about 22 weeks after the first antibodyexposure, about 23 weeks after the first antibody exposure, about 24weeks after the first antibody exposure, about 25 weeks after the firstantibody exposure, or about 26 weeks after the first antibody exposure.In some embodiments, the second antibody exposure is not provided untilless than about any of the following weeks after the first antibodyexposure: 26, 25, 24, 23, 22, 21, 20, or 19. In some embodiments, thesecond antibody exposure is not provided until greater than about any ofthe following weeks after the first antibody exposure: 18, 19, 20, 21,22, 23, 24, or 25. That is, the second antibody exposure is not provideduntil any of a range of weeks having an upper limit of 26, 25, 24, 23,22, 21, 20, or 19 and an independently selected lower limit of 18, 19,20, 21, 22, 23, 24, or 25, wherein the lower limit is less than theupper limit.

The dosing regimens described herein use a consistent system fortracking time between doses whereby the first dose is administered tothe patient on Day 1. As described herein, an antibody exposure of thepresent disclosure may include one or two doses. In cases where theantibody exposures contain one dose, references to a second antibodyexposure not provided until a period of time has elapsed after a firstantibody exposure (as described herein) refer to the amount of timeelapsed between the dose of the first antibody exposure (e.g., Day 1)and the dose of the second antibody exposure. If the first antibodyexposure includes two doses, the first dose of the first antibodyexposure is provided on Day 1. In cases where the antibody exposurescontain two doses, references to a second antibody exposure not provideduntil a period of time has elapsed after a first antibody exposure (asdescribed herein) refer to the amount of time elapsed between the firstof the two doses of the first antibody exposure (e.g., Day 1) and thefirst dose of the two doses of the second antibody exposure. Forexample, if a method of the present disclosure includes a first antibodyexposure with two doses and a second antibody exposure with two doses,and the second antibody exposure is not provided until about 22 weeksafter the first antibody exposure, then the interval between the firstdose of the first antibody exposure and the first dose of the secondantibody exposure is about 22 weeks.

In some embodiments, a first antibody exposure of the present disclosureincludes one or two doses of a type II anti-CD20 antibody of the presentdisclosure. In some embodiments, the first antibody exposure contains atotal exposure of between about 1800 mg and about 2200 mg of the type IIanti-CD20 antibody. In some embodiments, the first antibody exposurecontains a total exposure of about 1800 mg, about 1900 mg, about 2000mg, about 2100 mg, or about 2200 mg of the type II anti-CD20 antibody.

In some embodiments, the first antibody exposure includes two doses. Insome embodiments, the first antibody exposure includes a first dose ofbetween about 900 mg and about 1100 mg of the type II anti-CD20 antibodyand a second dose of between about 900 mg and about 1100 mg of the typeII anti-CD20 antibody. In some embodiments, the first dose of the firstantibody exposure contains about 1000 mg of the type II anti-CD20antibody. In some embodiments, the second dose of the first antibodyexposure contains about 1000 mg of the type II anti-CD20 antibody. Insome embodiments, the second dose of the first antibody exposure is notprovided until about 1.5 weeks to about 2.5 weeks after the first doseof the first antibody exposure. In some embodiments, the second dose ofthe first antibody exposure is not provided until about 2 weeks afterthe first dose of the first antibody exposure.

In some embodiments, a second antibody exposure of the presentdisclosure includes one or two doses of a type II anti-CD20 antibody ofthe present disclosure. In some embodiments, the second antibodyexposure contains a total exposure of between about 1800 mg and about2200 mg of the type II anti-CD20 antibody. In some embodiments, thesecond antibody exposure contains a total exposure of about 1800 mg,about 1900 mg, about 2000 mg, about 2100 mg, or about 2200 mg of thetype II anti-CD20 antibody.

In some embodiments, the second antibody exposure includes two doses. Insome embodiments, the second antibody exposure includes a first dose ofbetween about 900 mg and about 1100 mg of the type II anti-CD20 antibodyand a second dose of between about 900 mg and about 1100 mg of the typeII anti-CD20 antibody. In some embodiments, the first dose of the secondantibody exposure contains about 1000 mg of the type II anti-CD20antibody. In some embodiments, the second dose of the second antibodyexposure contains about 1000 mg of the type II anti-CD20 antibody. Insome embodiments, the second dose of the second antibody exposure is notprovided until about 1.5 weeks to about 2.5 weeks after the first doseof the second antibody exposure. In some embodiments, the second dose ofthe second antibody exposure is not provided until about 2 weeks afterthe first dose of the second antibody exposure.

In some embodiments, a type II anti-CD20 antibody of the presentdisclosure is administered intravenously (e.g., by IV infusion).

In some embodiments, the methods of the present disclosure furtherinclude administering an effective amount of an immunosuppressive agent(e.g., in conjunction with a type II anti-CD20 antibody as describedherein). Several classes of immunosuppressive agents are known in theart, including without limitation cytostatics (e.g., cytotoxic agentssuch as antibiotics, alkylating agents (e.g., cyclophosphamide, alsoknown as cytophosphane), inosine monophosphate dehydrogenase inhibitors,antimetabolites such as protein synthesis inhibitors, folic acidanalogs, purine analogs, pyrimidine analogs, and the like),immunosuppressive antibodies, glucocorticoids, drugs targetingimmunophilins (e.g., tacrolimus, sirolimus, rapamycin and analogsthereof, ciclosporin, and the like), mTOR active site inhibitors,mycophenolic acid and derivatives or salts thereof, TNF bindingproteins, interferons, opiods, and other small molecules (e.g.,fingolimod). In some embodiments, the immunosuppressive agent includesmycophenolic acid, a derivative of mycophenolic acid, or a salt ofmycophenolic acid. In some embodiments, the immunosuppressive agentincludes mycophenolate mofetil. In some embodiments, theimmunosuppressive agent includes CellCept® (Roche). In some embodiments,the immunosuppressive agent includes Myfortic® (Novartis). Effectiveamounts of the immunosuppressive agents of the present disclosure areknown in the art and readily ascertainable by standard assays. Forexample, mycophenolate mofetil may be administered at 2.0-2.5 g/day asillustrated in FIG. 1. In some embodiments, mycophenolate mofetil may beadministered starting at 1000 mg/day in divided doses (2 times/day) andtitrating up to 2.0-2.5 g/day in divided doses (2 times/day) by week 4.

In some embodiments, an immunosuppressive agent may be administeredbefore, during, or after administration of a type II anti-CD20 antibodyof the present disclosure, e.g., as a treatment for lupus. In someembodiments, an immunosuppressive agent may be administered throughoutthe period of treatment with a type II anti-CD20 antibody of the presentdisclosure. In some embodiments, mycophenolate mofetil may beadministered as described above throughout the period of treatment withthe type II anti-CD20 antibody.

In some embodiments, the methods of the present disclosure furtherinclude administering an effective amount of a glucocorticoid orcorticosteroid (e.g., in conjunction with a type II anti-CD20 antibodyas described herein). A variety of naturally occurring and syntheticglucocorticoids/corticosteroids are known in the art, including withoutlimitation beclometasone, triamcinolone, dexamethasone, betamethasone,prednisone, methylprednisolone, prednisolone, cortisone, and cortisol.In some embodiments, the glucocorticoids/corticosteroid includesmethylprednisolone. In some embodiments, theglucocorticoids/corticosteroid includes prednisone. Effective amounts ofthe glucocorticoids/corticosteroids of the present disclosure are knownin the art and readily ascertainable by standard assays. For example,methylprednisolone may be administered at 750-1000 mg doses once dailyby IV. As another example, prednisone may be administered orally at 0.5mg/kg and optionally tapered to 7.5 mg/day.

In some embodiments, a glucocorticoid may be administered before,during, or after administration of a type II anti-CD20 antibody of thepresent disclosure, e.g., to treat LN clinical activity. In someembodiments, a glucocorticoid may be administered prior toadministration of a type II anti-CD20 antibody of the presentdisclosure, e.g., 30-60 minutes before the type II anti-CD20 antibody.In some embodiments, 80 mg methylprednisolone may be administered by IV30-60 minutes before administration of a type II anti-CD20 antibody ofthe present disclosure. In some embodiments, prednisone (e.g., orallyadministered) and/or methyl prednisolone (e.g., IV administered) may beadministered with treatment, followed by a maintenance treatment (e.g.,mycophenolate mofetil or cyclophosphamide).

In some embodiments, the methods of the present disclosure furtherinclude administering an effective amount of an antihistamine (e.g., inconjunction with a type II anti-CD20 antibody as described herein).Antihistamines known in the art and currently in clinical use includehistamine H₁-receptor and histamine H₂-receptor antagonists or inverseagonists. In some embodiments, the antihistamine includesdiphenhydramine. Effective amounts of the antihistamines of the presentdisclosure are known in the art and readily ascertainable by standardassays. For example, diphenhydramine may be administered in 50 mg oraldoses.

In some embodiments, an antihistamine may be administered before,during, or after administration of a type II anti-CD20 antibody of thepresent disclosure, e.g., as a prophylactic treatment. In someembodiments, an antihistamine may be administered prior toadministration of a type II anti-CD20 antibody of the presentdisclosure, e.g., 30-60 minutes before the type II anti-CD20 antibody.In some embodiments, 50 mg diphenhydramine may be administered orally30-60 minutes before administration of a type II anti-CD20 antibody ofthe present disclosure.

In some embodiments, the methods of the present disclosure furtherinclude administering an effective amount of a non-steroidalanti-inflammatory drug or NSAID (e.g., in conjunction with a type IIanti-CD20 antibody as described herein). NSAIDs known in the art includeacetic acid derivatives, propionic acid derivatives, salicylates, enolicacid derivatives, anthranilic acid derivatives, selective COX-2inhibitors, sulfonanilides, and the like. In some embodiments, the NSAIDincludes acetaminophen. Effective amounts of the NSAIDs of the presentdisclosure are known in the art and readily ascertainable by standardassays. For example, acetaminophen may be administered in 650-1000 mgoral doses.

In some embodiments, an NSAID may be administered before, during, orafter administration of a type II anti-CD20 antibody of the presentdisclosure, e.g., as a prophylactic treatment. In some embodiments, anNSAID may be administered prior to administration of a type II anti-CD20antibody of the present disclosure, e.g., 30-60 minutes before the typeII anti-CD20 antibody. In some embodiments, 650-1000 mg acetaminophenmay be administered orally 30-60 minutes before administration of a typeII anti-CD20 antibody of the present disclosure.

In some embodiments, the methods of the present disclosure furtherinclude administering an effective amount of an anti-malarial agent(e.g., in conjunction with a type II anti-CD20 antibody as describedherein). Examples of anti-malarial agents that may be used includewithout limitation hydroxychloroquine, chloroquine, and quinacrine. Insome embodiments, an anti-malarial agent may be administered before,during, or after administration of a type II anti-CD20 antibody of thepresent disclosure, e.g., as a treatment for one or more symptoms oflupus.

In some embodiments, the methods of the present disclosure furtherinclude administering an effective amount of an integrin antagonist(e.g., in conjunction with a type II anti-CD20 antibody as describedherein). Examples of integrin antagonists that may be used includewithout limitation an LFA-1 antibody, such as efalizumab (RAPTIΓVA®)commercially available from Genentech, or an alpha 4 integrin antibodysuch as natalizumab (ANTEGREN®) available from Biogen, or diazacyclicphenylalanine derivatives, phenylalanine derivatives, phenylpropionicacid derivatives, enamine derivatives, propanoic acid derivatives,alkanoic acid derivatives, substituted phenyl derivatives, aromaticamine derivatives, ADAM disintegrin domain polypeptides, antibodies toalphavbeta3 integrin, aza-bridged bicyclic amino acid derivatives, etc.In some embodiments, an integrin antagonist may be administered before,during, or after administration of a type II anti-CD20 antibody of thepresent disclosure, e.g., as a treatment for one or more symptoms oflupus.

In some embodiments, the methods of the present disclosure furtherinclude administering an effective amount of a cytokine antagonist(e.g., in conjunction with a type II anti-CD20 antibody as describedherein). Examples of cytokine antagonists that may be used includewithout limitation an antagonist (e.g., an antagonist antibody) againstIL-1, IL-1α, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-15; a tumor necrosis factor such as TNF-α or TNF-β; and otherpolypeptide factors including LIF and kit ligand (KL). In someembodiments, a cytokine antagonist may be administered before, during,or after administration of a type II anti-CD20 antibody of the presentdisclosure, e.g., as a treatment for one or more symptoms of lupus.

In some embodiments, the methods of the present disclosure furtherinclude administering an effective amount of a hormone (e.g., inconjunction with a type II anti-CD20 antibody as described herein). Insome embodiments, a hormone (e.g., for hormone replacement therapy) maybe administered before, during, or after administration of a type IIanti-CD20 antibody of the present disclosure, e.g., for a medicaltreatment in a women with lupus.

In some embodiments, the methods of the present disclosure furtherinclude administering a standard of care treatment (e.g., in conjunctionwith a type II anti-CD20 antibody as described herein). In someembodiments, a standard of care treatment may be administered before,during, or after administration of a type II anti-CD20 antibody of thepresent disclosure, e.g., for treating or preventing one or moresymptoms of lupus. In certain embodiments, a standard of care treatmentmay be administered after a second antibody exposure of the presentdisclosure. For example, a type II anti-CD20 antibody of the presentdisclosure may be administered as described herein to a patient as aninduction therapy, then the patient may be treated according to standardof care as a maintenance therapy. Standard of care treatments for lupusare well known in the art and include without limitation anangiotensin-converting enzyme (ACE) inhibitor, an angiotensin-receptorblocker, cyclophosphamide, mycophenolate mofetil (e.g., at a dose asdescribed herein, such as 2.0-2.5 g/day), azathioprine, and aglucocorticoid or corticosteroid (e.g., prednisone, such as a prednisonetaper).

In some embodiments, the methods of the present disclosure furtherinclude administering an anti-hypertensive agent (e.g., in conjunctionwith a type II anti-CD20 antibody as described herein). In someembodiments, an anti-hypertensive agent may be administered before,during, or after administration of a type II anti-CD20 antibody of thepresent disclosure, e.g., for treating or preventing hypertension. Insome embodiments, anti-hypertensive agents includes without limitationACE inhibitors and angiotensin-receptor blockers. In some embodiments,an anti-hypertensive agent listed in Table 5 is administered, e.g., at adose within the ranges described in Table 5.

In some embodiments, the methods of the present disclosure result in acomplete renal response (CRR) in an individual. In some embodiments, aCRR comprises all of the following: a normalization of serum creatinine,an inactive urinary sediment, and a urinary protein to creatinine ratioof <0.5. In some embodiments, a normalization of serum creatinine ischaracterized by serum creatinine less than or equal to the upper limitof normal (ULN) range of central laboratory values, and/or serumcreatinine 15% above baseline and less than or equal to the ULN range ofcentral laboratory values if baseline (e.g., Day 1) serum creatinine iswithin the normal range of the central laboratory values. In someembodiments, an inactive urinary sediment is characterized by <10RBCs/high-power field (HPF) and/or the absence of red cell casts. Formore detailed discussion of CRR and partial renal response (PRR) in LN,see, e.g., Chen, Y. E. et al. (2008) Clin. J. Am. Soc. Nephrol. 3:46-53.

In some embodiments, the methods of the present disclosure result in acomplete renal response (CRR) or a partial renal response (PRR) in anindividual. In some embodiments, a PRR comprises one or more of thefollowing: a normalization of serum creatinine, an inactive urinarysediment, and a urinary protein to creatinine ratio of <0.5. In someembodiments, a PRR comprises one or more of the following: mitigation ofone or more symptoms including without limitation a reduction in serumcreatinine, reduced urinary sediment, a reduction in proteinuria, andany other improvement in renal function. In some embodiments, a CRR orPRR comprises a reduction in one or more biomarkers of lupus activity,including without limitation anti-dsDNA antibodies, antinuclearantibodies/ENA, anti-complement antibodies, reduced levels of complementC3 and/or C4, and reduced complement activity (e.g., as measured by CH50assay).

In some embodiments, the methods of the present disclosure result in adepletion of circulating peripheral B cells in an individual. In someembodiments, after administration of a type II anti-CD20 antibody of thepresent disclosure (e.g., according to any of the methods describedherein), circulating peripheral B cells are present in peripheral bloodat about 10 cells/μl, or fewer, about 9 cells/μl, or fewer, about 8cells/μl, or fewer, about 7 cells/μl, or fewer, about 6 cells/μl, orfewer, about 5 cells/μl, or fewer, about 4 cells/μl, or fewer, about 3cells/μl, or fewer, about 2 cells/μl, or fewer, or about 1 cell/μl, orfewer. In some embodiments, circulating peripheral B cells in theindividual are depleted by at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, orabout 100%. In some embodiments, depletion of circulating peripheral Bcells refers to a measurement of circulating peripheral B cells takenafter a first antibody exposure (e.g., including 1 or 2 doses of ananti-CD20 antibody as described herein), after a second antibodyexposure (e.g., including 1 or 2 doses of an anti-CD20 antibody asdescribed herein), 3 months after treatment (e.g., after receiving afirst and/or a second antibody exposure as described herein), 6 monthsafter treatment (e.g., after receiving a first and/or a second antibodyexposure as described herein), 9 months after treatment (e.g., afterreceiving a first and/or a second antibody exposure as describedherein), or 12 months after treatment (e.g., after receiving a firstand/or a second antibody exposure as described herein), e.g., ascompared to a corresponding measurement in the same individual beforetreatment, or as compared to a corresponding measurement in a controlindividual (e.g., an individual that has not received treatment).

Methods for assaying depletion of circulating peripheral B cells in anindividual are known in the art, e.g., flow cytometry using one or moreantibodies that recognize a B cell marker. In some embodiments, highlysensitive flow cytometry (HSFC) may be used to assay depletion ofcirculating peripheral B cells (see, e.g., Vital, E. M. et al. (2011)Arthritis Rheum. 63:3038-3047). In some embodiments, the B cells areCD19+ B cells. In some embodiments, the B cells are naïve B cells (e.g.,CD19+CD27− B cells), memory B cells (e.g., CD19+CD27+ B cells), orplasmablasts (e.g., CD19+CD27+CD38++ B cells).

IV. ARTICLES OF MANUFACTURE OR KITS

In another aspect of the invention, an article of manufacture or kitcontaining materials useful for the treatment, prevention and/ordiagnosis of the disorders described above is provided. The article ofmanufacture or kit comprises a container and a label or package inserton or associated with the container. Suitable containers include, forexample, bottles, vials, syringes, IV solution bags, etc. The containersmay be formed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is by itself or combined withanother composition effective for treating, preventing and/or diagnosingthe condition and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is an antibody described herein (e.g., a type IIanti-CD20 antibody of the present disclosure). The label or packageinsert indicates that the composition is used for treating the conditionof choice, e.g., according to any of the methods described herein.Alternatively, or additionally, the article of manufacture or kit mayfurther comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In some embodiments, provided herein is a kit comprising a containercomprising a type II anti-CD20 antibody of the present disclosure and anoptional pharmaceutically acceptable carrier, and, optionally, a packageinsert comprising instructions for treating or delaying progression oflupus nephritis in an individual, e.g., wherein the instructionsindicate that at least a first antibody exposure to a type II anti-CD20antibody and a second antibody exposure to the type II anti-CD20antibody are administered to the individual, the second antibodyexposure not being provided until from about 18 weeks to about 26 weeksafter the first antibody exposure; wherein the first antibody exposurecomprises one or two doses of the type II anti-CD20 antibody, the firstantibody exposure comprising a total exposure of between about 1800 mgand about 2200 mg of the type II anti-CD20 antibody; and wherein thesecond antibody exposure comprises one or two doses of the type IIanti-CD20 antibody, the second antibody exposure comprising a totalexposure of between about 1800 mg and about 2200 mg of the type IIanti-CD20 antibody. In some embodiments, provided herein is a kitcomprising a container comprising a type II anti-CD20 antibody of thepresent disclosure and an optional pharmaceutically acceptable carrier,and, optionally, a package insert comprising instructions for treatingor delaying progression of class III or class IV lupus nephritis in anindividual. In some embodiments of any of the above embodiments, thetype II anti-CD20 antibody comprises a heavy chain comprising HVR-H1sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ ID NO:2, and HVR-H3sequence of SEQ ID NO:3, and a light chain comprising HVR-L1 sequence ofSEQ ID NO:4, HVR-L2 sequence of SEQ ID NO:5, and HVR-L3 sequence of SEQID NO:6. In some embodiments of any of the above embodiments, the typeII anti-CD20 antibody is obinutuzumab.

The article of manufacture may still further comprise a second or thirdcontainer comprising a second medicament, wherein the anti-CD20 antibody(e.g., a type II anti-CD20 antibody of the present disclosure) is afirst medicament, where the article further comprises instructions onthe package insert for treating the subject with the second medicament.Exemplary second medicaments include a chemotherapeutic agent, animmunosuppressive agent, an anti-malarial agent, a cytotoxic agent, anintegrin antagonist, a cytokine antagonist, a hormone, and any of thetreatments that may be used in conjunction with a type II anti-CD20antibody as described herein. The article of manufacture in theseembodiments may further comprise a package insert indicating that thecompositions can be used to treat a particular condition.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-CD20 antibody.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1 A Pharmacology Study of Obinutuzumab Administered withMycophenolate Mofetil in Patients with Class III/IV Lupus NephritisStudy Design

This Phase II study is designed to assess the safety and efficacy ofobinutuzumab (i.e., a type II anti-CD20 antibody) as an add-on therapyto mycophenolate mofetil (MMF) in patients with active ISN/RPS ClassIII/IV lupus nephritis (LN). The Phase II study is a parallel-group,double-blind, randomized, placebo-controlled study comparing theefficacy and safety of obinutuzumab plus MMF with placebo plus MMF inClass III and IV patients with proliferative LN (FIG. 1).

The study is also a prospective, multicenter study. Patients diagnosedwith ISN/RPS Class III or IV LN, in some embodiments with a diagnosis ofSLE according to current ACR criteria (at least 4 criteria must bepresent, one of which must be a positive anti-nuclear antibody), areenrolled in centers throughout the world. The study includesstandard-of-care therapy with angiotensin-converting enzyme (ACE)inhibitors/angiotensin II receptor blockers, MMF (dosed at 2.0-2.5g/day), and a prednisone taper.

As described in greater detail below, patients are 18-75 years of ageand have ISN/RPS 2003 Class III or IV proliferative LN (see Weening, J J(2004) J. Am. Soc. Nephrol. 15:241-250) as evidenced by renal biopsyperformed within 6 months prior to screening and may have concomitantClass V disease (e.g., Class III/V or Class IV/V). Patients with ClassIII (C) or Class IV (C) disease are excluded because of the lowerlikelihood of response within these categories.

Inclusion criteria for the study include:

(a) Signed Informed Consent Form;

(b) Age 18-75 years;(c) Ability to comply with study protocol;(d) Diagnosis of systemic lupus erythematosus (SLE) according to currentACR criteria (at least 4 criteria must be present, one of which must bea positive anti-nuclear antibody);(e) Diagnosis of ISN/RPS 2003 Class III or IV LN as evidenced by renalbiopsy performed within 6 months prior to screening (patients may alsoco-exhibit Class V disease in addition to either Class III or Class IVdisease);(f) Demonstration of active urinary sediment as evidenced by 10 RBCs/HPFor the presence of red cell casts; and(g) Proteinuria (urine protein to creatinine ratio>1.0, based on a24-hour urine collection).

Key exclusion criteria include:

(a) Retinitis, poorly controlled seizure disorder, acute confusionalstate, myelitis, stroke or stroke syndrome, cerebellar ataxia, ordementia that is currently active and resulting from SLE;(b) Presence of rapidly progressive glomerulonephritis (defined by thepresence of crescent formation in 50% of glomeruli assessed on renalbiopsy or the doubling of serum creatinine within 12 weeks ofscreening);(c) Severe renal impairment as defined by estimated GFR<30 mL/min or theneed for dialysis or renal transplant;(d) Greater than 50% of glomeruli with sclerosis on renal biopsy;(e) Treatment with cyclophosphamide or calcineurin inhibitors within the3 months prior to randomization; and(f) Unstable disease with thrombocytopenia or at high risk fordeveloping clinically significant bleeding or organ dysfunctionrequiring therapies such as plasmapheresis or acute blood or platelettransfusions.

Patients receive an initial 1000 mg of methylprednisolone intravenously(IV) prior to or during screening, and may receive up to 3000 mgmethylprednisolone IV prior to randomization for severe clinicalactivity according to guidelines of routine care for these patients.Patients receive 80 mg methylprednisolone (or methylprednisoloneplacebo) IV on the day of the obinutuzumab/placebo infusion to reduceinfusion-related events. The oral prednisone taper is 0.5 mg/kg and isreduced over 12 weeks. This modified taper is initiated in recognitionthat prednisone doses above 10 mg/day are associated with significantadverse events, including increased risk of cardiovascular events(Bichile, T. and Petri, M. (2014) Presse Med. 43:e187-195). Priorexperience with rituximab suggests that it can potentially enablecomplete and partial renal responses in the absence of oral prednisoneor a prednisone taper, thus allowing the use of lower doses ofcorticosteroids (Condon, M. B. et al. (2013) Ann. Rheum. Dis.72:1280-1286).

Patients are followed for 12 months until the primary endpointevaluation, and an interim analysis at 6 months is performed to evaluateearly differences in CRR. All patients have central reading of the renalbiopsy histopathology and also receive repeat renal biopsy as availableon the basis of clinical status and local practice. All patients areevaluated by high sensitivity flow cytometry (HSFC) to evaluate theability of obinutuzumab to deplete circulating peripheral B cells, andan interim PD analysis is performed to assess whether patients do notfully deplete peripheral CD19+ B cells as anticipated.

Dosing and Non-Investigational Medicinal Products

The dosing regimen for the study is obinutuzumab administered by IVinfusion at a dose of 1000 mg on Days 1, 15, 168, and 182 (test group);or obinutuzumab placebo (e.g., saline IV, corresponding to theobinutuzumab1000-mg dose) administered by IV infusion on Days 1, 15,168, and 182. The obinutuzumab/placebo is administered in a hospital orclinic environment where full resuscitation facilities are immediatelyavailable and under close supervision of the investigator or designee.After the end of the infusion, the IV line remains in place for at least1 hour to enable administration of IV drugs if necessary. If no adverseevents occur during this period of time, the IV line may be removed.

After screening, patients who are not already receiving MMF receive 1500mg/day MMF in divided doses (2-3 times/day), and all patient doses aretitrated up to a target dose of 2.0-2.5 g/day in divided doses (2-3times/day) by Week 4, as tolerated. If reductions in dose are necessary,decreases are allowed in 250-500 mg decrements. During screening or atrandomization, if clinically indicated, patients may receive 750-1000 mgmethylprednisolone IV once daily for up to three days to treatunderlying LN clinical activity. Patients receive 0.5 mg/kg oralprednisone during screening or at randomization, tapering thisprednisone dose, per protocol, starting on Day 16 and reducing theprednisone dosage to 7.5 mg/day by Week 12. These treatments aredescribed in further detail below.

Concomitant Therapy and Clinical Practice

Patients who are not already taking vitamin D (400 IU/day) and calciumsupplements (1200 mg/day of calcium citrate or 1500 mg/day of calciumcarbonate) begin taking these supplements at randomization. All patientstake either an angiotensin-converting enzyme inhibitor or an angiotensinreceptor blocker titrated to adequate blood pressure control asrecommended by the National Kidney Foundation for chronic kidneydisease. Other agents that affect proteinuria are not allowed to beinitiated during the study, including but not limited tonon-dihydropyridine calcium antagonists, dihydropyridine calciumantagonists, aldosterone antagonists, and direct renin antagonists.

Mycophenolate Mofetil (MMF)

All patients continue on or initiate use of MMF during screening or nolater than Day 1. The initial dosage is 1500 mg/day by mouth, given intwo or three divided doses and titrated upward to 2.0-2.5 g/day individed doses by Week 4. MMF may be increased by 500 mg/week, astolerated up to a maximum dosage of 2.5 g/day. Reductions are allowedbecause of adverse effects.

Newly diagnosed patients with LN with no prior exposure to MMF arerecommended to initiate an induction agent (MMF or cyclophosphamide) andthen be re-assessed for eligibility. A proportion of patients who areinitially treated with MMF or cyclophosphamide achieve CRR and thereforehave minimal need for added immunosuppression (Dall'Era, M. et al.(2011) Arthritis Care Res. 63:351-357).

For those patients who enter the study already receiving a dosage of MMFhigher than 1500 mg/day, MMF will be titrated upward, as tolerated, to agoal of 2.5 g/day, given in divided doses, by Week 4. A patient'scurrent dose of MMF is given in 2 or 3 divided doses and increased by500 mg/week as tolerated.

Corticosteroid Administration

All patients receive a combination of IV and oral corticosteroids aspart of their initial therapy for LN. Methylprednisolone (e.g.,Solu-Medrol®) is implemented for two purposes: as part of the usual carefor patients with active Class III or IV LN and also to reduceinfusion-related reactions (IRRs) on the days of obinutuzumab/placeboinfusions. Up to three doses of IV methylprednisolone 1000 mg are givenon the basis of investigator judgement and local practice. Up to three1000 mg infusions may have been initiated prior to screening or duringthe screening interval.

On Days 1, 15, 168, and 186, patients receive 80 mg IVmethylprednisolone or placebo 30-60 minutes prior to study drug infusionto prevent IRRs. Additionally, oral prednisone may be initiated beforeor during the screening interval, and a taper commences on Day 2. FromDays 2 to 16, 0.50 mg/kg/day oral prednisone is given (maximum dose 60mg), except on the day of IV methylprednisolone/placebo infusions, andwill continue until Day 16. From Day 16 onward, a prednisone tapercommences.

All patients undergo a scheduled corticosteroid taper commencing on Day16. Patients fractionally reduce their prednisone dose over 12 weeksuntil the dose is 7.5 mg/day by Week 12. After 14 weeks of tapering,patients continue on prednisone at 7.5 mg/day or less. In patients whosedisease is too clinically active for the patient to make the first stepin their prednisone taper, as evidenced by active urinary sediment,rising serum creatinine, or moderate-to-severe extra-renal symptoms,these patients may continue to receive their initial prednisone dose forup to an additional 28 days.

To maintain consistency in the treatment of renal flares, retreatmentwith higher doses of corticosteroids is permitted if judged clinicallyappropriate by the investigator and if patients meet criteria for arenal flare. Patients may be treated with prednisone (up to 0.5 mg/kg;not to exceed 60 mg/day) for 2 weeks. Prednisone is then tapered toachieve 10 mg/day within 6 weeks after initial corticosteroid increase.Patients are allowed to receive corticosteroids for emergent illness(trauma, severe asthma) or surgery, if clinically warranted; thecorticosteroid use is limited to a total of ≦7 days, if possible.Investigators are then allowed to increase the prednisone dose by ≦2.5mg/day to treat symptoms of adrenal insufficiency or corticosteroidwithdrawal, after the patient's dosage has been tapered to 10 mg/day.

Patients who experience a severe extra-renal SLE flare may receivetreatment with additional oral corticosteroids, if judged clinicallyappropriate by the investigator. These patients may be retreated withprednisone (up to 1.0 mg/kg) for up to 2 weeks on the basis of theseverity of disease and organ system involvement and the dosage istapered to 7.5 mg/day. Patients experiencing a mild or moderateextra-renal flare may temporarily increase their prednisone dose by upto 20 mg per day and taper this dose over 4 weeks, if judged clinicallyappropriate by the investigator. IV corticosteroids in equivalent dosesare allowed if gastrointestinal involvement temporarily precludestreatment with oral corticosteroids.

Anti-Malarial Medications

Patients taking anti-malarial medications at study entry maintain aconstant dosage throughout the study. Patients not previously onanti-malarial medications may be enrolled in the study but should notinitiate anti-malarial medications unless experiencing a disease flarethat is unresponsive to corticosteroids. Table 4 lists anti-malarialmedications and dose ranges expected to be used during the course of thestudy.

TABLE 4 Anti-malarial medications Anti-malarial Medication Dose Range(oral) Hydroxychloroquine 200-400 mg daily Chloroquine 500 mg every dayor every other day Quinacrine 100 mg every day

Antihypertensive Therapy

All patients who are not currently taking either an ACE inhibitor or anangiotensin-receptor blocker should be started on one at screening.Patients are on either an ACE inhibitor or angiotensin-receptor blockerfor at least 10 days prior to randomization. Combination therapy withthe two agents is not allowed.

During screening, every effort is made to adequately control patients'blood pressures. The dose of the ACE inhibitor or angiotensin-receptorblocker may be titrated upward to the maximum recommended dose in thecurrent package insert to achieve adequate blood pressure control asrecommended by the Eighth Report of the Joint National Committee on thePrevention, Detection, Evaluation, and Treatment of High Blood Pressure(James, P. A. et al. (2014) JAMA 311:507-520). If adequate bloodpressure control is not achieved, patients may be started on additionalantihypertensive agents but not on agents that affect proteinuria (e.g.,nondihydropyridine calcium channel blockers, aldosterone antagonists,direct renin antagonists). Additional agents that specifically targetthe renin-angiotensin system are not initiated during the study.Suggested dose ranges for specific ACE inhibitors andangiotensin-receptor blockers are listed in Table 5. If patients areintolerant to ACE inhibitors and angiotensin-receptor blockers, they mayuse either a direct renin inhibitor or aldosterone antagonists, but notin combination.

TABLE 5 Suggested Dose Ranges for ACE Inhibitors andAngiotensin-Receptor Blockers ACE Inhibitor or Angiotensin- Dose Range(Oral), Receptor Blocker mg/day ACE Inhibitors Benazepril 10-80 Ramipril2.5-10  Lisinopril 10-80 Enalapril 10-40 Quinapril 10-80 Captopril 75-450 Perindopril  4-16 Trandolapril 1-8 Moexipril 7.5-30 Angiotensin-Receptor Blockers Eprosartan 400-600 Valsartan  80-320Olmesartan 20-40 Candesartan  8-32 Telmisartan 20-80 Losartan  25-100Irbesartan  75-300

Study Objectives and Outcome Measures

The primary objective of this proof-of-concept study is to measurecomplete renal response (CRR) at 52 weeks with administration ofobinutuzumab. The ability of obinutuzumab plus MMF to achieve a CRR atweek 52 is compared to placebo plus MMF and assessed by improvements inrenal function, urinary sediment, and proteinuria. Secondary objectivesinclude evaluations of the safety of obinutuzumab in this patientpopulation, the ability of obinutuzumab to induce an overall response(CRR+PRR) at Week 52, the ability of obinutuzumab to improvetime-to-response (CRR+PRR) over the course of 52 weeks, and the abilityof obinutuzumab to improve biomarkers of LN disease activity (e.g.,reduced anti-dsDNA antibody levels, increased C3 and C4 levels; see Tew,G. W. et al. (2010) Lupus 19:146-157).

The primary efficacy outcome measure is the proportion of subjects whoachieve a CRR, evaluated at 52 weeks. In this study, CRR is defined byattainment of all of the following:

(a) Normalization of serum creatinine as evidenced by the following:

-   -   (i) Serum creatinine less than or equal to the upper limit of        normal (ULN) range of central laboratory values if the baseline        (Day 1) is not within the normal range of the central laboratory        values; and    -   (ii) Serum creatinine≦15% above baseline and less than or equal        to the ULN range of central laboratory values if baseline        (Day 1) serum creatinine is within the normal range of the        central laboratory values;        (b) Inactive urinary sediment (as evidenced by <10        RBCs/high-power field (HPF) and the absence of red cell casts);        and        (c) Urinary protein to creatinine ratio<0.5.

Any patient who switches to rescue medication prior to Week 52 isconsidered a non-responder. The proportions of patients achieving CRRacross treatment groups is compared using a Cochrane-Mantel-Haenzel(CMH) test with race (Afro-Caribbean/African-American versus others) andregion (United States versus non-United States) as stratificationfactors. If the test result is in favor of the obinutuzumab group atα<0.1-level (one-sided), a shift toward better renal response associatedwith the obinutuzumab group is concluded.

The secondary efficacy outcome measures are the following:

(a) Proportional analysis of patients who achieve an overall response atWeek 52 (CRR+PRR);(b) Time to overall response (CRR+PRR) over the course of 52 weeks;(c) Percent reduction or increase from baseline and mean and medianassessments of biomarkers of LN disease activity (e.g., reduction inanti-dsDNA antibody levels, increased C3 and C4 levels);(d) Proportion of patients that achieve a PRR at week 52 as defined byattainment of all of the following:

(i) serum creatinine≦15% above baseline levels;

(ii) RBCs/HPF≦50% above baseline and no red cell casts;

(iii) 50% improvement in the urine protein to creatinine ratio, with oneof the following conditions met:

-   -   (A) if the baseline urine protein to creatinine ratio is ≦3.0,        then a urine protein to creatine ratio of <1.0;    -   (B) if the baseline protein to creatinine ratio is >3.0, then a        urine protein to creatinine ratio of <3.0;        (e) Proportion of patients who achieve a CRR at Week 24;        (f) Time to CRR, over the course of 52 weeks;        (g) Proportion of patients that achieve a modified CRR (mCRR1)        at Week 52 employing the primary-efficacy measure definition and        removing the urinary sediment analysis criteria

mCRR1 refers to attainment of normalization of serum creatinine asevidenced by the following:

-   -   (i) Serum creatinine less than or equal to the ULN range of        central laboratory values;    -   (ii) Serum creatinine≦15% above baseline and less than or equal        to the ULN range of central laboratory values if baseline        (Day 1) serum creatinine is within the normal range of the        central laboratory values;    -   (iii) Urinary protein to creatinine ratio<0.5;        (h) Proportion of patients that achieve a second CRR (mCRR2) at        Week 52 as defined by attainment of the following:

(i) Normalization of serum creatinine as evidenced by the following:

-   -   (A) Serum creatinine≦the ULN range of central laboratory values;    -   (B) Serum creatinine≦15% above baseline if baseline (Day 1)        serum creatinine is above the normal range of the central        laboratory values OR S≦the ULN range of central laboratory        values if baseline (Day 1) serum creatinine is within the normal        range of the central laboratory values;

(ii) Inactive urinary sediment (as evidenced by <10 RBCs/HPF and theabsence of red cell casts);

(iii) Urinary protein to creatinine ratio<0.5.

The pharmacodynamic (PD) objective is to compare changes in CD19+ Bcells in the peripheral blood following treatment with obinutuzumabversus placebo. Levels of circulating CD19+ B-cells are measured atscreening and Days 15, 28, 84, 168, 364, and 728.

The pharmacokinetic (PK) objectives are to characterize thepharmacokinetics of obinutuzumab in the LN population and to assesspotential PK interactions between obinutuzumab and concomitantmedications, including mycophenolate mofetil (MMF). Non-linearmixed-effects modeling (with software NONMEM) is used to analyze thedose-concentration-time data of obinutuzumab. The PK profile data isused to further develop a PK model, including the effect of majorcovariates (e.g., sex, race/ethnicity, weight, biochemical andhematological parameters at baseline, degree of underlying disease), onthe main parameters (e.g., clearance). Derivation of individual measuresof exposure, such as area under the concentration-time curve (AUC) andmaximum concentration observed (C_(max)) depend on the final PK modelused. Serum obinutuzumab is summarized (mean, minimum, maximum, SD, andgeometric mean) and reported.

The exploratory objectives for the study include evaluation of pre-doselevels of exploratory biomarkers (including but not limited to B-cellsubsets and levels of protein and/or mRNA in serum, blood, and urine)and potential associations with outcome, evaluation of changes inexploratory biomarkers (including but not limited to B-cell subsets andlevels of protein and/or mRNA in serum, blood, and urine) over time inpatients dosed with obinutuzumab versus placebo, evaluation of theoccurrence of extrarenal flares, evaluation of the impact of therapy onpatient and physician-reported outcomes, and assessment of renal biopsyhistopathology (e.g., for the presence of CD19+ B cells at the screeningand/or subsequent biopsies). The exploratory outcome measures include:

(a) levels of circulating B-cell subsets at Screen and Days 15, 28, 84,168, 364, and 728;(b) levels of exploratory biomarkers (including but not limited toB-cell subsets and levels of protein and/or mRNA in serum, blood, andurine) at Screen and Days 1, 15, 28, 84, 168, 252, 364, 532, and 728;(c) proportion of patients experiencing a Systemic Lupus ErythematosusDisease Activity Index (SLEDAI)-2K flare;(d) proportion of patients experiencing a renal flare over 52 weeks and104 weeks;(e) proportion of patients achieving CRR, mCRR1, mCRR2 at additionaltimepoints (including Week 12 and 36);(f) Physician's Global Assessment (visual analog scale captured inscreening, at the baseline visit, and at several timepoints during studyconduct); and(g) renal biopsy evaluations.

Laboratory, Biomarker, and Other Biological Samples

The following laboratory assessments are recorded during the study:

(a) Hematology: hemoglobin, hematocrit, RBC, mean corpuscular volume,mean corpuscular hemoglobin, WBC (absolute and differential), andquantitative platelet count;(b) Blood chemistry: AST/SGOT, ALT/SGPT, alkaline phosphatase, amylase,lipase, total protein, albumin, cholesterol, total bilirubin, urea, uricacid, creatinine, random glucose, potassium, sodium, chloride, calcium,phosphorus, lactic dehydrogenase, CPK, and triglycerides;(c) Urinalysis: dipstick for blood, nitrate, protein, and glucose andurine microscopy;(d) 24-hour urine collection (analyzed for total protein, totalcreatinine, and creatinine clearance) to be performed at randomizationand at Months 3, 6, 9, and 12;(e) Flow cytometry: B cell (including CD19, CD27, CD38, and IgD), T cell(CD3, CD4, CD8), and NK cells (CD16, CD56);(f) Autoantibody profile: anti-nuclear antibody (ANA), anti-dsDNA,anti-Sm, anti-RNP, anti-Ro, anti-La, and anti-C1q;(g) Anti-dsDNA antibody: measured by ELISA at all visits as part ofSLEDAI-2K assessment;(h) Quantitative immunoglobulin: total Ig levels including IgG, IgM, andIgA isotypes;

(i) Complement: C3, C4, and CH50;

(j) Antibody titers: antibody titers to common antigens (rubella,tetanus, influenza, S. pneumoniae); and(k) Pregnancy test: urine pregnancy test performed at screening andprior to each study drug infusion. Infusion is not administered unlesstest is negative. At all other timepoints, urine pregnancy test isperformed on the basis of menstrual history and pregnancy risk.

The following samples are sent for analysis: cells from blood and urinefor B-cell and lupus-related biomarkers (including but not limited toCD19+ B cells and mRNA associated with B-cell activity), serum and urinefor B-cell and lupus-related biomarkers (including but not limited toB-cell activating factor or BAFF), and renal biopsy slides forimmunohistopathology assessment.

Infusions

Prior to each infusion of either study drug or placebo, patients receiveprophylactic treatment with acetaminophen (650-1000 mg) anddiphenhydramine (50 mg; or equivalent dose of a similar agent) by mouth,given 30-60 minutes before the start of the infusion period. Thepatients who are receiving obinutuzumab receive 80 mg methylprednisoloneIV and patients who are receiving placebo receiveplacebo-methylprednisolone IV given 30-60 minutes before the start ofthe obinutuzumab/placebo infusion. If a patient experiences a mildinfusion-related reaction (IRR) that is deemed by the investigator to beclinically significant, the infusion rate should be reduced to half ofthe initial infusion rate (in compliance with non-Hodgkin's lymphomaprotocol infusion rates and schedules). After the reaction has resolved,the infusion should be kept at the reduced rate for an additional 30minutes. If the reduced rate is tolerated, then the infusion rate may beincreased to the next closest rate on the infusion schedule. Patientswho experience a severe IRR should have their infusion interruptedimmediately and should receive aggressive symptomatic treatment. Theinfusion should not be restarted until all of the symptoms havedisappeared. Upon restarting the infusion, the rate should be half ofthat which precipitated the reaction. Instructions for administration ofobinutuzumab infusions are provided in Table 6 below.

TABLE 6 Administration of obinutuzumab infusions. First Infusion (Day 1)Subsequent Infusions Begin infusion at an initial rate of 50 mg/hr. If apatient experienced an infusion reaction If no infusion reaction occurs,increase the during the prior infusion, start at the same infusion ratein 50-mg/hr increments every rate as the first infusion (50 mg/hr) and30 minutes to a maximum of 400 mg/hr. follow those directions as noted.If an infusion reaction develops, stop or slow If the patient toleratedthe prior infusion the infusion. Administer infusion-reaction well,begin infusion at a rate of 100 mg/hr. medications and supportive carein If no infusion reaction occurs, increase the accordance withinstitutional protocol. infusion rate in 100-mg/hr increments everyResume the infusion at a 50% reduction in 30 minutes to a maximum of 400mg/hr. rate (the rate being used at the time that the If an infusionreaction develops, stop or slow hypersensitivity or infusion-relatedreaction the infusion. Administer infusion-reaction occurred) if thereaction has resolved, medications and supportive care in accordancewith institutional protocol. Resume the infusion at a 50% reduction inrate (the rate being used at the time that the hypersensitivity orinfusion-related reaction occurred) if the reaction has resolved.

All renal biopsies and reports that are obtained as part of study entryare photomicrographed and sent to an online central reading portal foroversight of the histopathologic assessment performed by the local renalhistopathologist. An expert panel assesses these biopsies andadjudicates a final interpretation. Every effort is made to completethis process while patients are in screening but is not mandatory forcompletion of screening activities. All new biopsies obtained duringscreening or during the study are processed in a manner to enableimmunohistochemical staining of the tubulointerstitium for the presenceof B cells. The study encourages but does not mandate the performance ofrepeat renal biopsies for patients that have not achieved a CRR, andseeks to enrich for study centers that perform repeat renal biopsies.

Example 2 Obinutuzumab Outperforms Rituximab at Inducing B-CellCytotoxicity in Rheumatoid Arthritis and Systemic Lupus ErythematosusPatient Samples Through Fc Gamma Receptor-Dependent and IndependentEffector Mechanisms

A proportion of Rheumatoid Arthritis (RA) and Systemic LupusErythematosus (SLE) patients treated with standard doses of rituximab(RTX) display inefficient B cell deletion and poor clinical responseswhich can be augmented by delivering higher doses, indicating thatstandard-dose RTX is a sub-optimal therapy in these patients. Toinvestigate whether better responses could be achieved with otheranti-CD20 mAbs, a comparison was made between RTX with Obinutuzumab(OBZ), a new-generation, glycoengineered type II anti-CD20 mAb in aseries of in vitro assays measuring B cell cytotoxicity in SLE and RAsamples. It was found that OBZ was at least 2-fold more efficient thanRTX at inducing B-cell cytotoxicity in in-vitro whole blood assays.Dissecting this difference it was found that RTX elicited more potentcomplement-dependent cellular cytotoxicity (CDC) than OBZ. In contrast,OBZ was more effective at evoking Fc gamma receptor (FcγR)-mediatedeffector mechanisms including activation of NK cells and neutrophils.OBZ was also more efficient at inducing direct cell death. This was truefor all CD19+ B-cells as a whole and in naïve (IgD+CD27−); and switched(IgD-CD27+) memory B-cells specifically, a higher frequency of which isassociated with poor clinical response after RTX.

Materials and Methods Patients

All participants of this study provided consent according to thedeclaration of Helsinki approved by the local research ethics committee.All patients with RA satisfied the American College of Rheumatology(ACR)/European League Against Rheumatism classification criteria(Aletaha D. et al. 2010 Ann Rheum Dis. 2010; 69(9):1580-8) and allpatients with SLE met the ACR classification criteria (Petri M. et al.Arthritis Rheum. 2012; 64(8):2677-86).

Antibodies and Reagents

Anti-CD20 mAbs used in the studies included RTX, OBZ andnon-glycoengineered, wild type glycosylated OBZ (OBZ_(Gly)) and in someexperiments OBZ with a mutated Fc portion (P329G LALA) that does notengage any Fc related effector functions (Herter S. et al. CancerResearch. 2015; 75(15 Supplement):2460), OBZ-PG LALA. Roche InnovationCenter Zurich, Switzerland generated all anti-CD20 mAbs except RTX,which was a kind gift from the pharmacy of University College Hospital,U.K. AT10, an FcγRII antagonist (Greenman J. et al. Mol Immunol. 1991;28(11):1243-54), was produced in-house.

Flow Cytometry and B Cell Isolation

Fluorochrome-conjugated mAb were procured from Becton Dickinsonbiosciences or Biolegend, U.K.): CD3 (phycoerythrin [PE]-Cy 7), CD15(fluorescein isothiocyanate, FITC): CD16 (Allophycoyanin, APC), CD19(Alexa Fluor 700), CD45 (PE), CD56 (PE), CD 107a (Brilliant Violet 421),CD11b (PE), CD62L (APC), propidium iodide and Annexin V (FITC). Flowcytometry was performer using a Becton Dickinson LSR Fortessa cellanalyzer. Lymphocytes were identified based on forward- and side-scattercharacteristics. B cells were identified as CD19+ or CD20+, T cells asCD3+ and NK cells as CD3-56+. Neutrophils were identified based onforward- and side-scatter characteristics and CD15 positively. The meanfluorescent intensity (MFI) of CD11b and CD62L in samples incubated withmAbs was compared with that in samples incubated without antibodies.

In all experiments, peripheral blood mononuclear cells (PBMC) wereseparated from whole blood samples by Ficoll-Hypaque density gradientand B-cells were isolated from PBMC using EasySep™ Human B CellEnrichment Kit (Cambridge, U.K.).

Whole Blood B-Cell Depletion Assays

Briefly, 300 μl of freshly drawn whole blood anticoagulated with heparinwas incubated with or without mAbs at 1 μg/mL for 24 hours at 37° C. and5% CO₂. Samples were then stained with anti-CD3, anti-CD19 and anti-CD45before lysing red cells and analyzing on flow cytometer, as describedpreviously (Reddy V. et al. Arthritis & rheumatology. 2015;67(8):2046-55). The % B-cell depletion was calculated from theproportion of B cells to T cells remaining after treatment and definedas the cytotoxicity index (CTI) as described previously (Mossner E. etal. Blood. 2010; 115(22):4393-402, and Reddy V. et al. Arthritis &rheumatology. 2015; 67(8):2046-55).

Surface Fluorescence-Quenching Assays

Surface fluorescence-quenching assays were performed as describedpreviously (Beers S. A. et al. Blood. 2008; 112(10):4170-7, and Reddy V.et al. Arthritis & rheumatology. 2015; 67(8):2046-55) to assessinternalization of mAbs by B-cells. Isolated B-cells were incubated for6 hours with Alexa-488 conjugated mAbs at a concentration of 5 μg/mLbefore analyzing by flow cytometry.

Complement-dependent Cellular Cytotoxicity Assays

CDC assays were performed as previously described (Cragg M. S. et al.Blood. 2004; 103(7):2738-43). Isolated B cells were incubated with mAbsat a concentration of 10 μg/mL for 30 minutes at 37° C. and 5% CO₂.Samples were stained with fluorescence conjugated anti-CD19 antibodies,Annexin V (Av) and propidium iodide (PI) and the frequency ofCD19+Av+PI+ cells assessed by flow cytometry. Freshly collected normalhealthy human serum was used as a source of complement. To define theactivity relating to complement, part of the serum was heat inactivated(HIS) at 56° C. for 30 minutes. The ability of mAbs to activatecomplement and lyse target cells was assessed by the relative frequencyof CD19+Av+PI+ cells in samples incubated either with normal healthyserum or HIS.

Direct Cell Death

Isolated B-cells were incubated in RPMI supplemented with 10% heatinactivated foetal calf serum with or without mAbs at a concentration of10 μg/mL for 6 hours at 37° C. and 5% CO₂. The frequency ofCD19+Av+cells in samples with mAbs compared with that in samples withoutmAbs represented the ability of mAbs to induce direct cell death.

NK Cell Degranulation Assays

NK cell degranulation was assessed using samples from the whole bloodB-cell depletion assay by measuring the expression of CD107a or LAMP-1(a lysosome associated membrane protein 1), which is up regulated uponactivation of NK cells and correlates with NK cell mediated ADCC (AlterG. et al. J Immunol Methods. 2004; 294(1-2):15-22, and Aktas E. et al.Cell Immunol. 2009; 254(2):149-54). Therefore, the frequency ofCD3−56+107a+ NK cells in samples with mAbs were compared with that insamples incubated without mAbs. Activation of NK cells is associatedwith an increased activity of metalloproteinase, which cleaves CD16reducing its expression upon NK cell activation (Romee R. et al. Blood.2013; 121(18):3599-608). Therefore the extent of CD16 loss was also usedas an indirect measure of NK cell activation (Grzywacz B. et al.Leukemia. 2007; 21(2):356-9; author reply 9, and Bowles J. A. et al.Blood. 2006; 108(8):2648-54).

Neutrophil Activation Assays

Neutrophil activation was assessed in the whole blood assay by measuringincreases in CD11b or decreases in CD62L on CD15+neutrophils by flowcytometry (Golay J. et al. Blood. 2013; 122(20):3482-91, and Wittmann S.et al. Cytometry A. 2004; 57(1):53-62). The ability of mAbs to induceneutrophil activation was assessed by comparing the mean fluorescentintensity (MFI) of CD11b and CD62L on CD15+neutrophils in samplesincubated with or without mAbs.

Statistical Analysis

Data was analyzed using Graph Pad Prism Software version 5.0. MannWhitney test or Wilcoxon matched-pairs signed rank test were used tocompare between groups as appropriate. Spearman correlation coefficientwas used to analyze for correlation.

Results

Type II mAbs are More Efficient than Type I at Inducing B-CellCytotoxicity

To assess the effect of Type I and II mAbs on B cell cytotoxicity in RAand SLE samples, whole blood B-cell depletion assays were performed asdescribed previously (Reddy V. et al. Arthritis & rheumatology. 2015;67(8):2046-55). OBZ was >2-fold more efficient than RTX at deletingB-cells from patients with RA (n=31) and SLE (n=34) with bothnon-glycomodified OBZ_(Gly) and OBZ more efficient than RTX in allsamples tested (FIG. 2A). In both RA and SLE, the median CTI of OBZ wassignificantly greater than the CTI of OBZ_(Gly) and RTX and the CTI ofOBZ_(Gly) was significantly higher than the CTI of RTX in both RA andSLE. In RA, the median (interquartile range) CTI of RTX, OBZ_(Gly) andOBZ was 29 (13-50), 60 (47-70) and 67 (60-77), respectively and in SLEwas 19 (11-39), 40 (31-53) and 59 (52-70), respectively. Thus, in bothRA and SLE, there was a hierarchy of mAb-induced B cell depletion:RTX<OBZ_(Gly)<OBZ. The remarkable inter-sample variability in B-celldepletion, particularly with RTX was also noted. The superior efficiencyof OBZ_(Gly) (having a non-glycomodified Fc similar to RTX) suggeststhat its type II nature accounts for the difference between the twotypes of mAbs in the efficiency of B-cell depletion in the whole bloodassay; whereas the increased efficiency of OBZ compared to OBZ_(Gly) isattributable to afucosylation of the Fc portion.

B-Cells Internalize RTX More Rapidly than OBZ

Next, it was investigated whether the superior efficiency of type IImAbs in B cell depletion was consistent with their type II nature, andso it assessed whether B-cells from patients with RA and SLEinternalized RTX to a greater extent than OBZ. It was found that RTXinternalized more extensively than OBZ after 6 hours of incubation witha median (range) percentage of surface accessible RTX vs OBZ of: 55(51-57) versus 83 (81-84), respectively in RA (n=5); and 60 (49-77)versus 76 (70-80), respectively in SLE (n=8) (FIG. 2B). To assess therole of FcγRIIb in this internalization, experiments were performed inthe presence of the FcγRII-blocking mAb AT10, which partially inhibitedinternalization of RTX and to a smaller extent, OBZ (FIG. 2B), similarto previous observations using a non-glycomodified Type II antibodyvariant of OBZ (Reddy V. et al. Arthritis & rheumatology. 2015;67(8):2046-55).

RTX is More Efficient than OBZ at Inducing Complement-Dependent CellularCytotoxicity

The ability of these mAbs to elicit CDC was also investigated. It wasfound that the frequency of lysed B cells (CD19+Av+PI+) wassignificantly greater in samples incubated with RTX in the presence ofnormal healthy serum (NHS) compared to heat inactivated serum (HIS) witha median (range) difference of 10.9% (8.1−21) whereas the difference forOBZ was 4.8% (0.9−6.5) (FIG. 2C). The mean±SD fold increase in lysedcells in samples incubated with NHS vs HIS was 1.9±0.5 and 1.2±0.2 forRTX and OBZ, respectively (FIG. 2D). Thus, the data suggests that RTXwas superior to OBZ at evoking CDC.

OBZ is More Efficient than RTX at Activating NK Cells

These CDC results were consistent with the type I and II nature of themAbs but at odds with the superior efficiency of type II mAbs in thewhole blood assay. Next, the ability of the mAbs to elicit FcγR-mediatedeffector mechanisms was investigated; first assessing NK activation inthe whole blood B-cell depletion assay. Gating as indicated in FIG. 3E,allowed assessment of NK degranulation (CD107a increase) relative toexpression of CD16. The highest proportion of CD107a+NK (CD3-CD56+)cells was seen in the CD56+CD16− fraction (FIG. 3A-3G) suggesting thatdegranulating NK cells had down regulated CD16 as previously reported(Grzywacz B. et al. Leukemia. 2007; 21(2):356-9; author reply 9).

Having established these parameters, equivalent assays were performedcomparing RTX and OBZ. After 24 hours of incubation in the absence ofmAbs there was no significant difference in the frequency of NK cells,CD107a+NK cells, CD16+ NK cells or B-cells between patients with RA(n=18) and SLE (n=23) (FIG. 4A). However, the median (range) frequencyof CD3−CD56+CD107a+ activated NK cells was significantly higher insamples incubated with OBZ compared to RTX 5.1% (1.9−22) vs 2.8%(0.3−14) and 5.5% (0.6−12) vs 4.3% (1.2−8.9), respectively) but therewas significantly lower median (range) frequency of CD16+NK cells, inboth RA and SLE 69 (36-94) vs 89 (83-97) and 66 (42-91) vs 84 (61-95),respectively (FIG. 4B). Also, there was a significantly higherfold-increase in the frequency of CD3-CD56+CD107a+ NK cells in samplesincubated with OBZ compared to those incubated with RTX in SLE (FIG.4B). Furthermore, it was found that NK cell activation, as assessed byeither gain of CD107a or loss of CD16; or the fold increase in thefrequency of CD3−CD56+CD107a+ NK cells, was greater in RA compared toSLE (FIG. 4B). NK cell activation, as assessed by the frequency ofCD3-CD56+CD107a+NK cells by RTX and OBZ, correlated significantly withthat in samples incubated without mAbs with r²=0.89, p<0.05; r²=0.78,p<0.05, respectively, in RA (FIG. 4C) and r²=0.52, p<0.05; r²=0.36,p<0.05, respectively, in SLE (FIG. 4D). However, correlations werestronger in RA compared to SLE. Taken together, these data suggestdisease related defects in the activation of NK cells in SLE maycontribute to inefficient B-cell depletion noted in whole blooddepletion assays (FIG. 2A) and that baseline activation status of NKcells may influence response to activation by mAbs in both RA and SLE(FIGS. 4B and 4C).

It was next investigated whether differential activation of NK cells byRTX and OBZ was due to type I and type II characteristics and/or due tothe effect of Fc engineering using either OBZ with wild-typeglycosylation (OBZ_(Gly)) or completely lacking FcγR engagement (OBZ-PGLALA), consequently, less/in-efficient at inducing ADCC and CDC (HessellA. J. et al. Nature. 2007; 449(7158):101-4). Significant differenceswere not found in the frequency of CD3-CD56+CD107a+ or CD3-CD56+CD16+NKcells in samples incubated without mAbs compared to samples incubatedwith OBZ-PG LALA in both RA (n=6) and SLE (n=12) showing thatFcγR-engagement is essential as expected. In these samples also OBZ wasmore efficient than OBZ_(Gly) and RTX at depleting B cells in the wholeblood assay in both RA (n=18) and SLE (n=23) (FIG. 5A), but anincreasing hierarchy was noted in the frequency of, and fold-increasein, CD3-CD56+CD107a+ NK cells as follows: no mAbs=OBZ-PG LALA<RTX<OBZ(FIGS. 5B and 5C). The frequency of CD3-CD56+CD16+NK cells wassignificantly lower in samples incubated with OBZ when compared withother samples (FIG. 5D). The frequency of CD3-CD56+CD16+NK cells wasalso lower in samples incubated with OBZ_(Gly) compared to RTX in RA,but not SLE (FIG. 5D).

Thus, the ability of mAbs to up-regulate the expression of CD107a onCD3-CD56+ NK cells was greater in RA compared with SLE, such that themean fold difference in samples incubated with RTX, OBZ-PG LALA,OBZ_(Gly) and OBZ when compared with samples incubated without mAbs was1.2, 1.5, 1.9 and 3.1, respectively, in RA and 1.5, 0.8, 1.4 and 1.8,respectively, in SLE (FIG. 5C). Although the pattern of B-cell depletionachieved by mAbs in RA and SLE (FIG. 5A) was similar to the pattern ofNK cell activation by mAbs (FIGS. 5B and 5D) there was no directcorrelation between % B-cell depletion achieved by mAbs and thefrequency of CD3-CD56+CD107a+ NK cells in individual samples (data notshown).

OBZ is More Efficient than RTX at Activating Neutrophils

In addition to NK cells, neutrophils have also been proposed as mAbeffector cells (Golay J. et al. Blood. 2013; 122(20):3482-91).Therefore, next, the ability of mAbs to induce neutrophil activation wasassessed by measuring the expression of CD11b and CD62L, as describedpreviously (Wittmann S. et al. Cytometry A. 2004; 57(1):53-62) and shownin FIG. 9. CD11b forms part of the β integrin (Mac-1) complex andseveral genetic variants of this complex have been associated withlupus-related phagocytic defects (Bologna L. et al. J Immunol. 2011;186(6):3762-9). Upon neutrophil activation the surface expression ofCD11b is up regulated whereas the expression of the adhesion moleculeCD62L is down regulated (Golay J. et al. Blood. 2013; 122(20):3482-91,and Wittmann S. et al. Cytometry A. 2004; 57(1):53-62). It was foundthat the MFI of CD11b in samples incubated with mAbs was significantlyhigher in both RA (n=10) and SLE (n=22) (FIG. 6A) when compared withsamples incubated without mAbs. In both RA and SLE, significantcorrelations were noted between the MFI of CD11b in samples incubated inthe absence of mAbs and that in samples incubated with RTX (r²=0.81,0.82, respectively) whereas significant correlation for OBZ was noted inSLE (r²=0.81), but not RA (FIG. 6B). A hierarchy was also noted in theability of mAbs to up-regulate CD11b such that the MFI of CD11b waslower in samples incubated with RTX<OBZ_(Gly)<OBZ, as in the case of NKcell activation. Also, the MFI of CD62L was greater in samples incubatedwith RTX>OBZ_(Gly)>OBZ (FIG. 6C). In both RA and SLE, significantcorrelations were noted between the MFI of CD62L in samples incubated inthe absence of mAbs and that in samples incubated with RTX (r²=0.93,0.91, respectively) and OBZ (r²=0.64, 0.71, respectively) (FIG. 6D).Thus, the hierarchy of mAbs in their ability to activate neutrophils wasOBZ>OBZ_(Gly)>RTX. Taken together, these data suggested that type IImAbs are superior to RTX in activating neutrophils in the whole bloodassay for both RA and SLE samples. OBZ-PG LALA did not elicitsignificant changes for either marker in both RA (n=7) and SLE (n=12)compared to samples incubated in the absence of mAbs.

OBZ is More Efficient than RTX at Inducing Direct Cell Death

Next, direct cell death (DCD) was assessed, using the Annexin V assay asshown in FIG. 10. The ability of OBZ to induce direct cell death wasgreater than that of RTX for both CD19+ cells as a whole and also B-cellsubpopulations; IgD+CD27− naïve cells and IgD-CD27+ switched memorycells, FIG. 7A (RA, n=5 and SLE, n=4). The proportion of Annexin V+cells was highest for DN cells>IgD+CD27+ unswitched memorycells>IgD-CD27+ switched memory cells>IgD+CD27− naïve cells, even insamples incubated without mAbs. Nonetheless, OBZ was superior to RTX atinducing DCD.

B-Cell Subpopulations: Expression of CD20, FcγRIIb and Internalizationof mAbs

It was next investigated whether differences between B-cellsubpopulations in the expression of CD20, FcγRIIb and/or their abilityto internalize mAbs provided explanations for the differentialsensitivity to mAb-induced DCD. B-cell subpopulations displayed varyingability to internalize mAbs such that IgD-CD27+ switched memory cellsinternalized mAbs less than other B-cell subpopulations; and IgD+CD27+unswitched memory cells internalized mAbs to a greater extent than otherB-cell subpopulations. Antagonizing the effects of FcγRIIb with AT10significantly reduced internalization in both cases. When compared tonaïve and IgD-CD27+ switched memory cells, IgD+CD27+ unswitched memorycells had significantly greater expression of CD20 and FcγRIIb anddisplayed significantly greater ability to internalize mAbs whereasnaïve and IgD-CD27+ switched memory cells had significantly lowerexpression of CD20 and FcγRIIb and displayed significantly lower levelsof internalization. DN cells had remarkably variable levels ofexpression of CD20 and FcγRIIb, but internalized RTX to a significantlygreater extent than IgD-CD27+ switched memory cells. B cells from bothRA and SLE samples consistently displayed low levels of OBZinternalization. Taking these data together, there was no clearrelationship between the susceptibility of B-cell subpopulations tomAb-induced DCD and the ability to internalize mAbs or to express CD20or FcγRIIb.

Here, it was shown that Obinutuzumab, a type II anti-CD20 mAb with aglycomodified Fc demonstrated at least 2-fold greater potency atdeleting B-cells from whole blood samples of patients with both RA andSLE compared to the RTX. This increased activity of OBZ was affectedpredominantly through Fc gamma receptor (FcγR)-mediated effectormechanisms and DCD. In contrast, RTX recruited complement moreefficiently for CDC, but was rapidly internalized and significantly lessefficient at evoking ADCC and DCD. The subsequent analysis revealed thatthe expression of the CD20 target molecule was less on IgD-CD27+switched memory and DN cells; perhaps accounting for their relativeresistance to removal by RTX.

1. A method for treating or delaying progression of lupus nephritis inan individual that has lupus, comprising administering to the individualat least a first antibody exposure to a type II anti-CD20 antibody and asecond antibody exposure to the type II anti-CD20 antibody, the secondantibody exposure not being provided until from about 18 weeks to about26 weeks after the first antibody exposure; wherein the first antibodyexposure comprises one or two doses of the type II anti-CD20 antibody,the first antibody exposure comprising a total exposure of between about1800 mg and about 2200 mg of the type II anti-CD20 antibody; wherein thesecond antibody exposure comprises one or two doses of the type IIanti-CD20 antibody, the second antibody exposure comprising a totalexposure of between about 1800 mg and about 2200 mg of the type IIanti-CD20 antibody; and wherein the type II anti-CD20 antibody comprisesa heavy chain comprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequenceof SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chaincomprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ IDNO:5, and HVR-L3 sequence of SEQ ID NO:6.
 2. The method of claim 1,wherein the first antibody exposure comprises a first dose of betweenabout 900 mg and about 1100 mg of the type II anti-CD20 antibody and asecond dose of between about 900 mg and about 1100 mg of the type IIanti-CD20 antibody.
 3. The method of claim 1, wherein the first antibodyexposure comprises a first dose of the type II anti-CD20 antibody and asecond dose of the type II anti-CD20 antibody, and wherein the seconddose of the first antibody exposure is not provided until from about 1.5weeks to about 2.5 weeks after the first dose of the first antibodyexposure.
 4. The method of claim 3, wherein the first antibody exposurecomprises a first dose of the type II anti-CD20 antibody and a seconddose of the type II anti-CD20 antibody, and wherein the second dose ofthe first antibody exposure is not provided until about 2 weeks afterthe first dose of the first antibody exposure.
 5. The method of claim 3,wherein the first dose of the first antibody exposure is about 1000 mgof the type II anti-CD20 antibody.
 6. The method of claim 3, wherein thesecond dose of the first antibody exposure is about 1000 mg of the typeII anti-CD20 antibody.
 7. The method of claim 1, wherein the secondantibody exposure comprises a first dose of between about 900 mg andabout 1100 mg of the type II anti-CD20 antibody and a second dose ofbetween about 900 mg and about 1100 mg of the type II anti-CD20antibody.
 8. The method of claim 1, wherein the second antibody exposurecomprises a first dose of the type II anti-CD20 antibody and a seconddose of the type II anti-CD20 antibody, and wherein the second dose ofthe second antibody exposure is not provided until from about 1.5 weeksto about 2.5 weeks after the first dose of the second antibody exposure.9. The method of claim 8, wherein the second antibody exposure comprisesa first dose of the type II anti-CD20 antibody and a second dose of thetype II anti-CD20 antibody, and wherein the second dose of the secondantibody exposure is not provided until about 2 weeks after the firstdose of the second antibody exposure.
 10. The method of claim 8, whereinthe first dose of the second antibody exposure is about 1000 mg of thetype II anti-CD20 antibody.
 11. The method of claim 8, wherein thesecond dose of the second antibody exposure is about 1000 mg of the typeII anti-CD20 antibody.
 12. The method of claim 1, wherein the firstantibody exposure and the second antibody exposure are administeredintravenously.
 13. The method of claim 1, wherein the individual hasclass III or class IV lupus nephritis.
 14. The method of claim 1,wherein the individual is at risk for developing class III or class IVlupus nephritis.
 15. A method for treating or delaying progression oflupus nephritis in an individual that has lupus, comprisingadministering to the individual an effective amount of a type IIanti-CD20 antibody; wherein the type II anti-CD20 antibody comprises aheavy chain comprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequenceof SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chaincomprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ IDNO:5, and HVR-L3 sequence of SEQ ID NO:6; and wherein the individual hasclass III or class IV lupus nephritis.
 16. The method of claim 14,wherein the type II anti-CD20 antibody is administered intravenously.17. The method of claim 1, wherein the individual does not have classIII (C) or class IV (C) lupus nephritis.
 18. The method of claim 1,wherein the individual has class V lupus nephritis.
 19. The method ofclaim 1, further comprising administering to the individual an effectiveamount of an immunosuppressive agent.
 20. The method of claim 19,wherein the immunosuppressive agent comprises mycophenolic acid, aderivative thereof, or a salt thereof.
 21. The method of claim 20,wherein the immunosuppressive agent comprises mycophenolate mofetil. 22.The method of claim 1, further comprising administering to theindividual an effective amount of a glucocorticoid or corticosteroid.23. The method of claim 22, wherein the glucocorticoid or corticosteroidcomprises methylprednisolone.
 24. The method of claim 22, wherein theglucocorticoid or corticosteroid comprises prednisone.
 25. The method ofclaim 1, further comprising administering to the individual an effectiveamount of an antihistamine.
 26. The method of claim 25, wherein theantihistamine comprises diphenhydramine.
 27. The method of claim 1,further comprising administering to the individual an effective amountof a non-steroidal anti-inflammatory drug (NSAID).
 28. The method ofclaim 27, wherein the NSAID comprises acetaminophen.
 29. The method ofclaim 1, further comprising administering to the individual an effectiveamount of an antihypertensive agent.
 30. The method of claim 29, whereinthe antihypertensive agent is an angiotensin-converting enzyme (ACE)inhibitor or an angiotensin-receptor blocker.
 31. The method of claim 1,further comprising administering to the individual a standard of caretreatment.
 32. The method of claim 31, wherein the standard of caretreatment comprises treatment with one or more of anangiotensin-converting enzyme (ACE) inhibitor, an angiotensin-receptorblocker, cyclophosphamide, mycophenolate mofetil, azathioprine, and aglucocorticoid or corticosteroid.
 33. The method of claim 1, wherein themethod results in a complete renal response (CRR) in the individual. 34.The method of claim 1, wherein the method results in a depletion ofcirculating peripheral B cells in the individual.
 35. The method ofclaim 34, wherein the circulating peripheral B cells are CD19+ B cells.36. The method of claim 1, wherein the type II anti-CD20 antibody is ahumanized or human antibody.
 37. The method of claim 1, wherein the typeII anti-CD20 antibody is afucosylated.
 38. The method of claim 1,wherein the heavy chain of the type II anti-CD20 antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:7.
 39. The method of claim 1, wherein the light chain of the type IIanti-CD20 antibody comprises a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:8.
 40. The method of claim 1,wherein the type II anti-CD20 antibody is obinutuzumab.
 41. The methodof claim 1, wherein the individual is a human.
 42. A kit for treating ordelaying progression of lupus nephritis in an individual that has lupus,comprising: (a) a container comprising a type II anti-CD20 antibody,wherein the type II anti-CD20 antibody comprises a heavy chaincomprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ IDNO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chain comprisingHVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ ID NO:5, andHVR-L3 sequence of SEQ ID NO:6; and (b) a package insert withinstructions for treating or delaying progression of lupus nephritis inan individual, wherein the instructions indicate that at least a firstantibody exposure to a type II anti-CD20 antibody and a second antibodyexposure to the type II anti-CD20 antibody are administered to theindividual, the second antibody exposure not being provided until fromabout 18 weeks to about 26 weeks after the first antibody exposure;wherein the first antibody exposure comprises one or two doses of thetype II anti-CD20 antibody, the first antibody exposure comprising atotal exposure of between about 1800 mg and about 2200 mg of the type IIanti-CD20 antibody; and wherein the second antibody exposure comprisesone or two doses of the type II anti-CD20 antibody, the second antibodyexposure comprising a total exposure of between about 1800 mg and about2200 mg of the type II anti-CD20 antibody.
 43. The kit of claim 42,further comprising a container comprising: (c) a second medicament,wherein the type II anti-CD20 antibody is a first medicament; and (d)instructions on the package insert for administering the secondmedicament to the subject.
 44. The kit of claim 43, wherein the secondmedicament is an immunosuppressive agent, a glucocorticoid, acorticosteroid, an anti-malarial agent, a cytotoxic agent, an integrinantagonist, a cytokine antagonist, or a hormone.
 45. A kit for treatingor delaying progression of lupus nephritis in an individual that haslupus, comprising: (a) a container comprising a type II anti-CD20antibody, wherein the type II anti-CD20 antibody comprises a heavy chaincomprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ IDNO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chain comprisingHVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ ID NO:5, andHVR-L3 sequence of SEQ ID NO:6; and (b) a package insert withinstructions for treating or delaying progression of class III or classIV lupus nephritis in an individual.
 46. The kit of claim 45, furthercomprising a container comprising: (c) a second medicament, wherein thetype II anti-CD20 antibody is a first medicament; and (d) instructionson the package insert for administering the second medicament to thesubject.
 47. The kit of claim 46, wherein the second medicament is animmunosuppressive agent, a glucocorticoid, a corticosteroid, ananti-malarial agent, a cytotoxic agent, an integrin antagonist, acytokine antagonist, or a hormone.
 48. A method for treating or delayingprogression of rheumatoid arthritis (RA) or systemic lupus erythematosus(SLE) in an individual, comprising administering to the individual aneffective amount of an anti-CD20 antibody, wherein the antibodycomprises a heavy chain variable region comprising an HVR-H1 sequence ofSEQ ID NO:1, an HVR-H2 sequence of SEQ ID NO:2, and an HVR-H3 sequenceof SEQ ID NO:3, and a light chain variable region comprising an HVR-L1sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and anHVR-L3 sequence of SEQ ID NO:6.
 49. The method of claim 48, wherein theantibody is administered intravenously.
 50. The method of claim 48,wherein the method results in a depletion of circulating peripheral Bcells in the individual.
 51. The method of claim 50, wherein thecirculating peripheral B cells are CD19+ B cells.
 52. The method ofclaim 48, wherein the antibody is a humanized or human antibody.
 53. Themethod of claim 48, wherein the antibody is afucosylated.
 54. The methodof claim 48, wherein the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:7.
 55. The method of claim 48, wherein thelight chain variable region comprises the amino acid sequence of SEQ IDNO:8.
 56. The method of claim 48, wherein the antibody is obinutuzumab.57. The method of claim 48, wherein the antibody comprises a modified Fcregion.
 58. The method of claim 57, wherein the Fc region comprises amodification for attenuating effector function.
 59. The method of claim57, wherein the Fc region is a human IgG1 Fc region.
 60. The method ofclaim 59, wherein the Fc region comprises L234A, L235A and P329G aminoacid substitutions, numbering according to EU index.
 61. The method ofclaim 48, wherein the individual is a human.